Methods and Compositions for Treatment of Disorders and Diseases Involving RDH12

ABSTRACT

Codon optimized nucleic acid sequences for RDH12 are provided, as well as recombinant viral vectors, such as AAV, expression cassettes, proviral plasmids or other plasmids containing the codon optimized sequence for functional RDH12. Recombinant vectors are provided that express the codon optimized, functional RDH12. Compositions containing these codon optimized sequences are useful in methods for treating, retarding or halting certain blinding diseases resulting from the absence, deficiency or inappropriate expression of RDH12. Other compositions and methods are providing for correcting a non-functional, defective or inadequately expressed native RDH12.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. Nonprovisional patentapplication Ser. No. 17/523,492, filed Nov. 10, 2021, which is acontinuation of U.S. Nonprovisional patent application Ser. No.16/314,179, filed Dec. 28, 2018, now U.S. Pat. No. 11,197, 936, issuedDec. 14, 2021, which is a national stage entry of International PatentApplication No. PCT/US2017/041122, filed Jul, 7, 2017, and which claimsthe benefit of the priority of U.S. Provisional patent application Ser.Nos. 62/359,777, filed Jul. 8, 2016, the contents of each of which areherein incorporated by reference in their entirety.

BACKGROUND OF THE INVENTION

Retinoid dehydrogenases/reductases located in the photoreceptor cellsand the RPE catalyze important oxidation-reduction reactions in thevisual cycle. RDH12 retinol dehydrogenase 12 (all-trans/9-cis/11-cis)(14q23.3-q24.1) (MIM no. 608830) encodes a dual specificity enzymeexpressed in the retina that acts on both trans and cis retinoidsubstrates. See, e.g., Thompson, DA et al, Nov. 2005, Human Mol. Genet.,14(24):3865-3875. A number of mutations in human RDH12 have beenassociated with certain forms of severe, childhood-onset autosomalrecessive retinal dystrophy (arRD). For example, defects in this geneare a cause of Leber congenital amaurosis type 13 and RetinitisPigmentosa 53.

Leber's Congenital Amaurosis (LCA), a severe dystrophy of the retina,affects about 1 in 80,000 people worldwide. The disorder is autosomalrecessive and carriers present with abnormal development ofphotoreceptors at birth or within the first few months of life.Abnormalities result in severe vision impairment and blindness.RDH12-associated LCA is one of the rarer forms of LCA. Only about 2.7%of the LCA cases are caused by a mutation in RDH12.

Retinitis pigmentosa (RP) 53 is another retinal dystrophy belonging tothe group of pigmentary retinopathies. Retinitis pigmentosa ischaracterized by retinal pigment deposits visible on fundus examinationand primary loss of rod photoreceptor cells followed by secondary lossof cone photoreceptors. Patients typically have night vision blindnessand loss of midperipheral visual field. As their condition progresses,they lose their far peripheral visual field and eventually centralvision as well. RDI-12 is also associated with this blinding disease.

Current treatment of these blinding diseases is primarily supportive,involving referral of patients to programs for the visually impairedchild, correction of refractive error and use of low-vision aids, whenpossible, and access to occupational/educational therapies. Periodicallyaffected patients receive ophthalmic evaluation and, in those withresidual vision, are examined for the presence of amblyopia, glaucoma,or cataract, with appropriate therapies. Gene augmentation therapies arebeing evaluated for the treatment of certain forms of Leber's congenitalamaurosis (LCA). See, e.g., U.S. Pat. No. 8,147,823.

A continuing need in the art exists for new and effective tools andmethods for successful treatment of LCA13, RP53 and other oculardiseases.

SUMMARY OF THE INVENTION

Therapeutic compositions and methods useful in the treatment of oculardisorders involve novel cDNA sequences optimized to encode functionalRDH12 protein and the delivery of the optimized sequences to a subjectwith an ocular disease. In one embodiment, such novel sequences includeplasmid sequences capable of being packaged in viral vectors. In otherembodiments, novel AAV proviral plasmids and/or recombinant AAV areprovided to carry the optimized sequences. As discussed herein, thesevectors have demonstrated biological activities both in vitro and invivo.

In one aspect, a codon optimized cDNA sequence encoding a functionalmammalian, preferably human, RDH12 is provided.

In another aspect an expression cassette comprises a codon optimizednucleic acid sequence that encodes one or more functional copies ofRDH12. In one embodiment, an expression cassette further comprises acodon optimized nucleic acid sequence that encodes one or morefunctional copies of RDH12 operably linked and under the control ofregulatory sequences directing its expression in a host cell, positionedbetween 5′ and 3′ AAV ITR sequences.

In other aspects, a vector is provided that contains one or more of theexpression cassettes described herein and host cells are provided thatcontain the vectors or expression cassettes.

In another aspect, a proviral plasmid comprises sequences encoding anAAV capsid, AAV inverted terminal repeat sequences and an expressioncassette comprising a codon optimized nucleic acid sequence that encodesnative or mutated or codon optimized RDH12 and expression controlsequences that direct expression of the encoded protein in a host cell.In certain embodiments, the plasmid components are modular.

In another embodiment, a recombinant adeno-associated virus (AAV)comprises an AAV capsid protein and a nucleic acid sequence encoding afunctional RDH12 protein, or fragment thereof, under the control ofregulatory sequences which express the RDH12 in the photoreceptor cellsof a subject. In one embodiment, the rAAV comprises an AAV8 capsid orvariant thereof, or an AAV7 capsid or variant thereof, or an AAV2 capsidor variant thereof, or an AAVS capsid or variant thereof.

In another embodiment, an rAAV comprises AAV inverted terminal repeatsequences and an expression cassette comprising a codon optimizednucleic acid sequence that encodes functional RDH12 and expressioncontrol sequences that direct expression of the encoded protein in ahost cell.

In yet a further aspect a pharmaceutical composition comprises apharmaceutically acceptable carrier, diluent, excipient and/or adjuvantand the optimized nucleic acid sequence encoding functional RDH12, and aplasmid, a vector, or a viral vector, such as the rAAV, describedspecifically herein. In one embodiment, the optimized nucleic acidsequence is under the control of regulatory sequences which express thefunctional

RDH12 in the photoreceptor cells of a subject. In one embodiment, thecomposition contains multiple copies of sequences encoding functionalRDH12. In another embodiment, the composition includes apharmaceutically acceptable carrier.

In a further aspect, a cell is provided that expresses functional RDH12protein encoded by a codon-optimized RDH12 cDNA. In one embodiment, sucha cell is an induced pluripotent stem cell (iPSC) which has treated bygene editing systems such as Crispr/Cas9 system and related systems andcomponents to target and correct mutations in the cell's existing RDH12sequences or to insert RDH12 codon optimized sequences for novelexpression in that cell.

In another aspect, a method of treating an RDH12-mediated disorder in amammalian subject comprises administering to a subject in need thereofthe codon-optimized RDH12 cDNA described herein, or a vector, virus orpharmaceutical compositions as described herein.

In another aspect, a method for treating, retarding or haltingprogression of blindness, or restoring at least partial vision, in amammalian subject comprises administering or delivering to the subjectan optimized nucleic acid sequence or fragment thereof encoding afunctional RDH12 protein, or fragment thereof. In one embodiment, themethod utilizes any of the compositions described herein.

In another aspect, a method for treating, retarding or haltingprogression of blindness or restoring at least partial vision in amammalian subject comprises administering a recombinant adeno-associatedvirus (AAV) comprising an AAV capsid protein and a nucleic acid sequenceencoding a functional RDH12 protein, or fragment thereof, under thecontrol of regulatory sequences which express the RDH12 in thephotoreceptor cells of a subject and a pharmaceutically acceptablecarrier. In one embodiment, the method utilizes any of the compositionsdescribed herein.

In still another aspect, a method to treat an ocular disorder resultingfrom expression of non-functional or improperly functional RDH12 orinadequate amounts or deficiencies of RDH12 comprises administering to asubject with the deficiency a vector comprising a native orcodon-optimized RDH12 gene under the control of a suitable promoter.

In still another aspect, a method to treat RDH12 deficiency or RDH12mutation-associated disorder comprises employing gene editing systemssuch as Crispr/Cas9 system and related systems and components to targetand correct mutations in RDH12. In one embodiment, the correctioninvolves optimizing the RDH12 encoding sequence in vivo.

In another embodiment, a method of treating or preventing LCA or RPcaused by a defect, deficiency or mutation in RDH12 in a subject in needthereof is provided. The method includes (a) identifying a subjecthaving, or at risk of developing, RDH12-associated LCA or RP; (b)performing genotypic analysis and identifying a mutation in the RDH12gene; (c) performing non-invasive retinal imaging and functional studiesand identifying areas of retained photoreceptors that could be targetedfor therapy; (d) administering to the subject an effective concentrationof a composition comprising a vector (e.g., a recombinant virus)carrying a nucleic acid sequence encoding a functional RDH12 under thecontrol of a promoter sequence which expresses the product of saidsequence in said photoreceptor cells, and a pharmaceutically acceptablecarrier, wherein said disorder is prevented, arrested or ameliorated.

Still other aspects and advantages of these compositions and methods aredescribed further in the following detailed description of the preferredembodiments thereof

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A-1B are an alignment of codon optimized RDH12 (SEQ ID NO: 5; topsequence) with native RDH12 (SEQ ID NO: 1; bottom sequence) showingabout 78% sequence similarity. In this alignment, the score is 787 bits(872), the identities are 744/949 (78%); the gaps are 0/949 (0%); andthe two strands are plus strands.

FIGS. 2A-2F are 6 panels of showing the expression of the RDH12 plasmid:left (FIG. 2A) and right (FIG. 2B) top panels show untransfectedcontrol; left (FIG. 2C) and right (FIG. 2D) middle panels show RDH12Myctransfected cells; left bottom panel shows RDH12Myc transfected cells(FIG. 2E); right bottom panel (FIG. 2F) is an enlargement of two cellsfrom the right middle panel (FIG. 2D).

FIG. 2G is a gel showing RDH12Myc transfected COS-7 cells, control COS 7cells, RDH12Myc transfected CHO cells and control Cho cells and twomolecular weight markers.

FIG. 3 is a gel showing expression of AAV2-RDH12Myc in RDH12 iPs cells.The cells are labeled and MW markers shown.

FIG. 4 is a graph showing A-wave prebleach and post bleach ratio inRDH12.myc injected vs. uninjected retinal of RDH12 KO mice withAAV8-RDH12-myc and AAV7m8RDH12-myc. Electroretinograms (ERG) wereperformed on RDH12 −/− mice that were injected in one eye with our testvector. Contralateral uninjected eye was used as a control to comparethe protective effect of exogenous RDh12 over light induced retinaldamage. ERG were performed before and after light damage to compare theaffect. Post light damage, Uninjected eyes showed a decrease in A-waveamplitudes, while the injected eyes remained relatively stable afterlight damage.

FIG. 5A shows a graph of an A-wave prebleach and post-bleach ratio inRDH12.myc (AAV8-RDH12-Myc) injected vs. uninjected retina of RDH12 KOmice.

FIG. 5B shows a graph of an A-wave prebleach and post-bleach ratio inRDH12.myc (AAV7m8-RDH12-Myc) injected vs. uninjected retina of RDH12 KOmice.

FIGS. 6A-6D show experimental results for a single animal 136 in whichthe left eye (FIGS. 6A AND 6C) was uninjected. The right eye (FIGS. 6Band 6D) was injected with AAV7m8-RDH12-Myc. ERG baseline was performedfollowed by light damage, followed by a second ERG. The animals werehoused for 10 days and a third ERG was performed. Mice were sacrificedand eyes collected fixed and sectioned and stained with DAPI (FIG. 6Aand 6B) or with rhodopsin and DAPI (FIG. 6C and 6D).

FIG. 7A through 7C show that retinal architecture is preserved in anAAV7m8-RDH12-Myc injected retina compared to uninjected retina afterlight damage. FIG. 7A shows the left uninjected eye. FIG. 7B shows ahigher magnification image of the left eye, showing a thin ONL. FIG. 7Cis a right eye injected with AAV7m8-RDH12-Myc.

FIG. 8A shows the retinal architecture of an animal with a left eyeuninjected showing a thin retina. FIG. 8B shows the animal's right eye,injected with AAV8-RDH12-Myc.

FIGS. 9A and 9B show the retinal architecture of a single animal 147with higher magnification images. FIG. 9A shows uninjected left eye.FIG. 9B shows right eye injected with AAV8-RDH12-Myc.

FIG. 10A is a schematic map of pAAV.CBAe.h-Native-RDH12, an expressioncassette which contains between an AAV 5′ ITR and 3′ITR, the nativenucleic acid sequence encoding functional RDH12 under the control ofregulatory sequences including the CMV.CβA promoter (CBAe), directingexpression of the RDH12 in a selected cell.

FIG. 10B is a schematic map of pAAV.CBAe.h-Native-RDH12.myc, anexpression cassette which contains between an AAV 5′ ITR and 3′ITR, thenative nucleic acid sequence encoding functional RDH12 linked to a myctag under the control of regulatory sequences including the CMV.CβApromoter directing expression of the RDH12 in a selected cell.

FIG. 10C is a schematic map of pAAV.CBAe.h-cocon opt-RDH12, anexpression cassette which contains between an AAV 5′ ITR and 3′ITR, thecodon optimized nucleic acid sequence encoding functional RDH12 (SEQ IDNO: 3) linked to a myc tag under the control of regulatory sequencesincluding the CMV.CβA promoter directing expression of the RDH12 in aselected cell.

DETAILED DESCRIPTION

The methods and compositions described herein are useful for thetreatment of ocular disorders. Such ocular disorders are RDH12-mediateddisorders or diseases, e.g., disorders, caused by, or involving amutation, defect or deficiency in the gene encoding human retinaldehydrogenase 12 (RDH12). In one embodiment, these compositions andmethods are useful in delivering a codon optimized cDNA encodingfunctional human retinal dehydrogenase gene (hRDH12) to mammaliansubjects for the treatment of ocular disorders. In certain embodiments,the RDH12-mediated disorder is a blinding disease such as LCA or LCA13,or RP or RP53. In other embodiments, the methods and compositions areuseful in editing the subject's defective gene in vivo or in creating asuitable cell line expressing the codon optimized cDNA sequence encodingfunctional RDH12 using gene editing systems such as the CRISPR/Cassystem. The compositions and methods described herein involve expressioncassettes, vectors, recombinant viruses and other compositions fordelivery of one or multiple, different versions of the sequence encodinga functional RDH12. Such compositions involve both codon optimizationand the assembly of multiple and or different versions of RDH12 in thesame expression cassette, vector or virus. These features not onlyincrease the efficacy of the functional RDH12 protein being expressed,but may also permit a lower dose of a therapeutic reagent that deliversthe functional protein to increase safety. It is anticipated thatoptimization of the nucleic acid sequence encoding RDH12 delivered in acassette or virus maximizes the level of production of functional RDH12protein in vivo, compared to levels that can be generated using thenative or endogenous sequence encoding RDH12.

Technical and scientific terms used herein have the same meaning ascommonly understood by one of ordinary skill in the art to which thisinvention belongs and by reference to published texts, which provide oneskilled in the art with a general guide to many of the terms used in thepresent application. The definitions contained in this specification areprovided for clarity in describing the components and compositionsherein and are not intended to limit the claimed invention.

The terms “a” or “an” refers to one or more. For example, “an expressioncassette” is understood to represent one or more such cassettes. Assuch, the terms “a” (or “an”), “one or more,” and “at least one” areused interchangeably herein.

As used herein, the term “about” means a variability of plus or minus10% from the reference given, unless otherwise specified.

The words “comprise”, “comprises”, and “comprising” are to beinterpreted inclusively rather than exclusively, i.e., to include otherunspecified components or process steps. The words “consist”,“consisting”, and its variants, are to be interpreted exclusively,rather than inclusively, i.e., to exclude components or steps notspecifically recited.

“RDH12” is retinol dehydrogenase 12 (all-trans/9-cis/11-cis) protein,preferably the human ortholog thereof. This protein is also known asRP53, LCA13 and SDR7C2. The term “RDH12” as used herein, refers to thefull length protein itself or a functional fragment, or variant thereof,as further defined below. In one embodiment, the RDH12 protein sequenceis derived from the same mammal that the composition is intended totreat. In one embodiment, the RDH12 is derived from a human. In anotherembodiment, the RDH12 is derived from a canine.

By the term “fragment” or “functional fragment” when applied to aprotein, refers to any fragment that retains the function of the fulllength protein, although not necessarily at the same level of expressionor activity.

By “functional protein” is meant any amino acid sequence thatdemonstrates the biological activity of a normally functioning protein,e.g., RDH12, in a subject with no ocular disorder or disease. Such afunctional protein can carry mutations or modifications of its encodingDNA sequence or within its amino acid sequence, which mutations do notcause ocular disease or disorders. In an other embodiment, such afunctional protein can include mutations or which cause the protein toperform its functions better than a “native” or endogenous sequence. Inone embodiment, such a functional RDH12 protein can be considered anormal or normally-functioning protein.

In one embodiment, a native human RDH12 has the following sequencelabeled SEQ ID NO: 2 (which is the encoded protein of the nucleic acidsequence SEQ ID NO: 1, reported in the section B below):

MLVTLGLLTSFFSFLYMVAPSIRKFFAGGVCRTNVQLPGKVVVITGANTGIGKETARELASRGARVYIACRDVLKGESAASEIRVDTKNSQVLVRKLDLSDTKSIRAFAEGFLAEEKQLHILINNAGVMMCPYSKTADGFETHLGVNHLGHFLLTYLLLERLKVSAPARVVNVSSVAHHIGKIPFHDLQSEKRYSRGFAYCHSKLANVLFTRELAKRLQGTGVTTYAVHPGVVRSELVRHSSLLCLLWRLFSPFVKTAREGAQTSLHCALAEGLEPLSGKYFSDCKRTWVSPRARNNKTAERLWNVSCELLGIRWE.

In another embodiment, the RDH12 protein sequence is a variant whichshares at least 70, at least 75%, at least 78% or at least 80% identitywith a native RDH12 protein, such as SEQ ID NO: 1. In anotherembodiment, the RDH12 protein sequence shares at least 85% identity witha native RDH12 protein. In another embodiment, the RDH12 proteinsequence shares at least 90% identity with a native RDH12 protein. Inanother embodiment, the RDH12 protein sequence shares at least 91%identity with a native RDH12 protein. In another embodiment, the RDH12protein sequence shares at least 92% identity with a native RDH12protein. In another embodiment, the RDH12 protein sequence shares atleast 93% identity with a native RDH12 protein. In another embodiment,the RDH12 protein sequence shares at least 94% identity with a nativeRDH12 protein. In another embodiment, the RDH12 protein sequence sharesat least 95% identity with a native RDH12 protein. In anotherembodiment, the RDH12 protein sequence shares at least 96% identity witha native RDH12 protein. In another embodiment, the RDH12 proteinsequence shares at least 97% identity with a native RDH12 protein. Inanother embodiment, the RDH12 protein sequence shares at least 98%identity with a native RDH12 protein. In another embodiment, the RDH12protein sequence shares at least 99% identity with a native RDH12protein.

The terms “percent (%) identity”, “sequence identity”, “percent sequenceidentity”, or “percent identical” in the context of amino acid sequencesrefers to the residues in the two sequences which are the same whenaligned for correspondence. Percent identity may be readily determinedfor amino acid sequences over the full-length of a protein, polypeptide,about 70 amino acids to about 300 amino acids, or a peptide fragmentthereof or the corresponding nucleic acid sequence coding sequencers. Asuitable amino acid fragment may be at least about 8 amino acids inlength, and may be up to about 150 amino acids. Generally, whenreferring to “identity”, “homology”, or “similarity” between twodifferent sequences, “identity”, “homology” or “similarity” isdetermined in reference to “aligned” sequences. “Aligned” sequences or“alignments” refer to multiple nucleic acid sequences or protein (aminoacids) sequences, often containing corrections for missing or additionalbases or amino acids as compared to a reference sequence. Alignments areperformed using any of a variety of publicly or commercially availableMultiple Sequence Alignment Programs. Sequence alignment programs areavailable for amino acid sequences, e.g., the “Clustal X”, “MAP”,“PIMA”, “MSA”, “BLOCKMAKER”, “MEME”, and “Match-Box” programs.Generally, any of these programs are used at default settings, althoughone of skill in the art can alter these settings as needed.Alternatively, one of skill in the art can utilize another method whichprovides at least the level of identity or alignment as that provided bythe referenced methods. See, e.g., J. D. Thomson et al, Nucl. Acids.Res., “A comprehensive comparison of multiple sequence alignments”,27(13):2682-2690 (1999).

By “optimized” or “codon-optimized” RDH12 protein is meant an RDH12protein sequence encoded by a DNA sequence which differs from the nativeor naturally occurring sequence, such as that of SEQ ID NO: 1, by codonchanges that make silent, conservative or non-conservative amino acidchanges, or amino acid insertions or deletions in the protein. Thesechanges may increase protein production and/or enhance proteinconfirmation and stability. An optimized RDH12 protein as describedherein is a functional RDH12 protein encoded by a codon-optimized DNAsequence.

One embodiment of an optimized RDH12 protein is the translatedfunctional RDH12 protein sequence labeled SEQ ID NO: 4 (which is theencoded protein of the nucleic acid sequence SEQ ID NO: 3, reported inthe section B below):

AAATMLVTLGLLTSFFSFLYMVAPSIRKFFAGGVCRTNVQLPGKVVVITGANTGIGKETARELASRGARVYIACRDVLKGESAASEIRVDTKNSQVLVRKLDLSDTKSIRAFAEGFLAEEKQLHILINNAGVMMCPYSKTADGFETHLGVNHLGHFLLTYLLLERLKVSAPARVVNVSSVAHHIGKIPFHDLQSEKRYSRGFAYCHSKLANVLFTRELAKRLQGTGVTTYAVHPGVVRSELVRHSSLLCLLWRLFSPFVKTAREGAQTSLHCALAEGLEPLSGKYFSDCKRTWVSPRARNNKTAERLWNVSCELLGIRWE.

As above synonymous codon changes or codon changes resulting inconservative amino acid changes, or insertions or deletions can increaseprotein production and/or enhance protein confirmation and stability.Such optimization is useful for development of therapeutic reagents thatmaximize the level of production of the experimental protein compared tolevels that can be generated using the endogenous or native sequence.

The nucleic acid sequence encoding a normal or normally funcationalRDH12 protein may be derived from any mammal which natively expressesthe RDH12 protein, or homolog thereof. In one embodiment, a nativenucleic acid sequence encoding human RDH12, is reported at NCBI databaseaccession No. NC_000014.9.

In one embodiment, a native human RDH12 DNA sequence is labeled SEQ IDNO: 1:

ATGCTGGTCACCTTGGGACTGCTCACCTCCTTCTTCTCGTTCCTGTATATGGTAGCTCCATCCATCAGGAAGTTCTTTGCTGGTGGAGTGTGTAGAACAAATGTGCAGCTTCCTGGCAAGGTAGTGGTGATCACTGGCGCCAACACGGGCATTGGCAAGGAGACGGCCAGAGAGCTCGCTAGCCGAGGAGCCCGAGTCTATATTGCCTGCAGAGATGTACTGAAGGGGGAGTCTGCTGCCAGTGAAATCCGAGTGGATACAAAGAACTCCCAGGTGCTGGTGCGGAAATTGGACCTATCCGACACCAAATCTATCCGAGCCTTTGCTGAGGGCTTTCTGGCAGAGGAAAAGCAGCTCCATATTCTGATCAACAATGCGGGAGTAATGATGTGTCCATATTCCAAGACAGCTGATGGCTTTGAAACCCACCTGGGAGTCAACCACCTGGGCCACTTCCTCCTCACCTACCTGCTCCTGGAGCGGCTAAAGGTGTCTGCCCCTGCACGGGTGGTTAATGTGTCCTCGGTGGCTCACCACATTGGCAAGATTCCCTTCCACGACCTCCAGAGCGAGAAGCGCTACAGCAGGGGTTTTGCCTATTGCCACAGCAAGCTGGCCAATGTGCTTTTTACTCGTGAGCTGGCCAAGAGGCTCCAAGGCACCGGGGTCACCACCTACGCAGTGCACCCAGGCGTCGTCCGCTCTGAGCTGGTCCGGCACTCCTCCCTGCTCTGCCTGCTCTGGCGGCTCTTCTCCCCCTTTGTCAAGACGGCACGGGAGGGGGCGCAGACCAGCCTGCACTGCGCCCTGGCTGAGGGCCTGGAGCCCCTGAGTGGCAAGTACTTCAGTGACTGCAAGAGGACCTGGGTGTCTCCAAGGGCCCGAAATAACAAAACAGCTGAGCGCCTATGGAATGTCAGCTGTGAGCTTC TAGGAATCCGGTGGGAGT

In other embodiments, certain modifications are made to the RDH12 codingsequence in order to enhance the expression in the target cell. Suchmodifications include codon optimization, (see, e.g., U.S. Pat. Nos.7,561,972; 7,561,973; and 7,888,112, incorporated herein by reference)and conversion of the sequence surrounding the translational start siteto a consensus Kozak sequence: gccRccATGR. See, Kozak et al, NucleicAcids Res. 15 (20): 8125-8148, incorporated herein by reference. Acodon-optimized nucleic acid sequence differs from the native ornaturally occurring sequence, such as that of SEQ ID NO: 1, bysynonymous codon changes that increase protein production, expressionand/or enhance protein confirmation and stability.

Codon-optimized coding regions can be designed by various differentmethods. This optimization may be performed using methods which areavailable on-line, published methods, or a company which provides codonoptimizing services. One codon optimizing method is described, e.g., inInternational Patent Application Pub. No. WO 2015/012924, which isincorporated by reference herein. Briefly, the nucleic acid sequenceencoding the product is modified with synonymous codon sequences.Suitably, the entire length of the open reading frame (ORF) for theproduct is modified. However, in some embodiments, only a fragment ofthe ORF may be altered. By using one of these methods, one can apply thefrequencies to any given polypeptide sequence, and produce a nucleicacid fragment of a codon-optimized coding region which encodes thepolypeptide. Such optimization is useful for development of gene therapyreagents that maximize the level of production of the experimentalprotein compared to levels that can be generated using the endogenous ornative sequence. Such codon-optimized sequences may in one embodimentincrease the efficacy of the resulting therapeutic reagent compositions,but also permit a lower dose of reagent to be used, thus increasingtherapuetic safety.

A number of options are available for performing the actual changes tothe codons or for synthesizing the codon-optimized coding regionsdesigned as described herein. Such modifications or synthesis can beperformed using standard and routine molecular biological manipulationswell known to those of ordinary skill in the art. In one approach, aseries of complementary oligonucleotide pairs of 80-90 nucleotides eachin length and spanning the length of the desired sequence aresynthesized by standard methods. These oligonucleotide pairs aresynthesized such that upon annealing, they form double strandedfragments of 80-90 base pairs, containing cohesive ends, e.g., eacholigonucleotide in the pair is synthesized to extend 3, 4, 5, 6, 7, 8,9, 10, or more bases beyond the region that is complementary to theother oligonucleotide in the pair. The single-stranded ends of each pairof oligonucleotides are designed to anneal with the single-stranded endof another pair of oligonucleotides. The oligonucleotide pairs areallowed to anneal, and approximately five to six of thesedouble-stranded fragments are then allowed to anneal together via thecohesive single stranded ends, and then they ligated together and clonedinto a standard bacterial cloning vector, for example, a TOPO® vectoravailable from Invitrogen Corporation, Carlsbad, Calif. The construct isthen sequenced by standard methods. Several of these constructsconsisting of 5 to 6 fragments of 80 to 90 base pair fragments ligatedtogether, i.e., fragments of about 500 base pairs, are prepared, suchthat the entire desired sequence is represented in a series of plasmidconstructs. The inserts of these plasmids are then cut with appropriaterestriction enzymes and ligated together to form the final construct.The final construct is then cloned into a standard bacterial cloningvector, and sequenced. Additional methods would be immediately apparentto the skilled artisan. In addition, gene synthesis is readily availablecommercially.

One embodiment of a codon-optimized RDH12 DNA sequence is the RDH12 DNAsequence labeled SEQ ID NO: 3.

GCGGCCGCCACCATGTTGGTCACCCTCGGACTCCTTACCTCATTTTTCTCCTTCCTGTACATGGTCGCCCCGAGCATTAGAAAGTTCTTCGCCGGCGGAGTGTGTAGGACTAACGTGCAGTTGCCCGGGAAGGTCGTGGTGATTACTGGCGCCAACACTGGTATCGGAAAGGAAACTGCGCGGGAACTGGCGTCCAGAGGTGCCCGCGTGTACATTGCATGCCGCGACGTGCTGAAGGGAGAATCCGCCGCGTCCGAGATCCGGGTGGACACCAAAAATAGCCAGGTGCTCGTGCGGAAGCTGGATCTGTCCGACACCAAGTCAATCAGGGCCTTTGCCGAGGGGTTCCTGGCTGAAGAGAAGCAGCTCCACATTCTGATCAACAACGCCGGGGTCATGATGTGCCCCTACTCAAAGACCGCAGACGGCTTCGAAACCCACCTGGGCGTGAACCATCTGGGACACTTCCTGCTGACCTATCTGCTGCTGGAGCGACTGAAAGTGTCGGCTCCTGCTCGGGTCGTGAACGTGTCCAGCGTGGCCCATCACATCGGAAAGATCCCATTCCACGATCTCCAATCCGAGAAGCGGTACAGCAGGGGCTTCGCGTACTGTCACTCGAAGTTGGCCAACGTGCTCTTTACCCGCGAACTGGCCAAGCGGCTGCAGGGCACTGGCGTGACCACTTACGCCGTGCACCCTGGTGTCGTGCGGTCCGAGCTGGTCCGCCATTCCTCTCTTCTGTGCCTCCTGTGGAGACTCTTCTCCCCGTTCGTCAAGACCGCAAGGGAAGGAGCCCAAACGAGCCTTCACTGTGCCCTGGCGGAAGGACTGGAGCCGCTTAGCGGAAAGTACTTCTCGGACTGCAAGCGCACCTGGGTGTCGCCTAGAGCTCGGAACAACAAGACTGCCGAACGCCTCTGGAATGTGTCCTGCGAGCTGCTGGGAATCAGATGGGAGTGATGATCATGAGATCT

When aligned with the native nucleic acid sequence encoding human RDH12,a codon optimized RDH12-encoding sequence may have a percent identity ofat least 50%, or at least 60%, or at least 70%, or at least 80% or atleast 90%, including any integer between any of those ranges. In oneembodiment, the codon optimized RDH12 has a percent identify with thenative sequence of at least 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61,62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79,80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97,98 or 99%.

The terms “percent (%) identity”, “sequence identity”, “percent sequenceidentity”, or “percent identical” in the context of nucleic acidsequences refers to the bases in the two sequences which are the samewhen aligned for correspondence. The percent identity is determined bycomparing two sequences aligned under optimal conditions over thesequences to be compared. The length of sequence identity comparison maybe over the full-length of the RDH12 coding sequence, or a fragment ofat least about 100 to 150 nucleotides, or as desired. However, identityamong smaller fragments, e.g. of at least about nine nucleotides,usually at least about 20 to 24 nucleotides, at least about 28 to 32nucleotides, at least about 36 or more nucleotides, may also be desired.Multiple sequence alignment programs are also available for nucleic acidsequences. Examples of such programs include, “Clustal W”, “CAP SequenceAssembly”, “BLAST”, “MAP”, and “MEME”, which are accessible through WebServers on the internet. Other sources for such programs are known tothose of skill in the art. Alternatively, Vector NTI utilities are alsoused. There are also a number of methods known in the art that can beused to measure nucleotide sequence identity, including those containedin the programs described above. As another example, polynucleotidesequences can be compared using Fasta™, a program in GCG Version 6.1.Commonly available sequence analysis software, more specifically, BLASTor analysis tools provided by public databases may also be used.

In the embodiment of SEQ ID NO: 3, the cDNA of a native human RDH12 wasmodified by the inventors by adding a complete Kozak consensus at the 5′end embedded in a NotI site and by adding WI and BamHI sites at the 3′end (restriction sites for cloning). A TGA stop codon was embedded inthe MI site to facilitate optimal epitope tagging. This embodiment alsoavoids the use of certain internal restriction enzymes such as Bg1II,Bsu36I, NheI, NotI, Sa1I and XhoI. A NotI restriction site with aKozak-CACC sequence is inserted at nucleotide 1-12 of SEQ ID NO: 3. ABg1II restriction site is inserted at the last six nucleotides of SEQ IDNO: 3.

Specifically FIGS. 1A-1B show a sequence alignment of SEQ ID NO: 5(codon-optimized RDH12 DNA sequence) with SEQ ID NO: 1 (native RDH12coding sequence). The open reading frame of codon optimized RDH12differs from the native sequence by 22% of the nucleotides (i.e. 78%homology), although the resulting encoded protein is the same.

As used herein, the term “subject” as used herein means a mammaliananimal, including a human, a veterinary or farm animal, a domesticanimal or pet, and animals normally used for clinical research. In oneembodiment, the subject of these methods and compositions is a human.Still other suitable subjects include, without limitation, murine, rat,canine, feline, porcine, bovine, ovine, and others. As used herein, theterm “subject” is used interchangeably with “patient”. The subjectincludes any mammal in need of these methods of treatment orprophylaxis, including particularly humans. The subject may be male orfemale. In one embodiment, the subject has, or is at risk of developing,Leber congenital amaurosis (LCA) or retinitis pigmentosa. In anotherembodiment, the subject has or is at risk of developing LCA or RP orother ocular disorder associated with a mutation in, or lack of, orinadequately expressed functional RDH12. In another embodiment, thesubject has shown clinical signs of LCA or RP. Clinical signs of LCA orRP include, but are not limited to, nystagmus, decreased peripheralvision, decreased central (reading) vision, decreased night vision, lossof color perception, reduction in visual acuity, decreased photoreceptorfunction, pigmentary changes and blindness. In another embodiment, thesubject has been diagnosed with LCA or RP. In yet another embodiment,the subject has not yet shown clinical signs of LCA or RP.

In yet another embodiment, the subject has about or at least 5 to 10%photoreceptor damage and/or loss. In another embodiment, the subject hasat least 20% photoreceptor damage and/or loss. In another embodiment,the subject has at least 30% photoreceptor damage and/or loss. Inanother embodiment, the subject has at least 40%, at least 50%, at least60% photoreceptor damage/loss. In another embodiment, the subject has atleast 70%, at least 80%, or at least 90% photoreceptor damage and/orloss. In one another embodiment, the subject has about or at least 5 to10% or more rod and/or cone function damage/loss. In one anotherembodiment, the subject has at least 20% rod and/or cone functiondamage/loss. In one another embodiment, the subject has at least 30% rodand/or cone function damage/loss. In one another embodiment, the subjecthas at least 40% rod and/or cone function damage/loss. In one anotherembodiment, the subject has at least 50% rod and/or cone functiondamage/loss. In one another embodiment, the subject has at least 60%, atleast 70%, at least 80% rod and/or cone function damage/loss. In oneanother embodiment, the subject has has 90% rod and/or cone functiondamage/loss.

As used herein, the term “disorder” or “genetic disorder” or“RDH12-mediated disorder” is used throughout this specific to refer toany diseases, disorders, or conditions associated with an insertion,change or deletion in the amino acid sequence of the native (e.g.,wild-type) RDH12 protein which renders the RDH12 protein eitherpartially or wholly non-functional in the subject's ocular cells. Thedisorder or genetic disease can also involve some other defect whichrenders the RDH12 protein either partially or wholly non-functional orpartially or wholly expressed in the subject's ocular cells. Unlessotherwise specified such disorders include inherited and/ornon-inherited genetic disorders, as well as diseases and conditionswhich may not manifest physical symptoms during infancy or childhood.

As used herein, the term “ocular cells” refers to any cell in, orassociated with the function of, the eye. The term may refer to any oneor more of photoreceptor cells, including rod, cone and photosensitiveganglion cells, retinal pigment epithelium (RPE) cells, Mueller cells,bipolar cells, horizontal cells, amacrine cells. In one embodiment, theocular cells are the photoreceptor cells. In another embodiment, theocular cells are the rod and cone cells. In yet another embodiment, theocular cells are the cone cells.

In certain embodiments of this invention, a subject has an “oculardisorder”, for which the components, compositions and methods of thisinvention are designed to treat. As used herein “ocular disorder”includes, rod-cone dystrophies and retinal diseases including, withoutlimitation, Stargardt disease (autosomal dominant or autosomalrecessive), retinitis pigmentosa, age-related macular degeneration,rod-cone dystrophy, Leber's congenital amaurosis, Usher's syndrome,Bardet-Biedl Syndrome, Best disease, Bassen-Kornzweig syndrome,retinoschisis, untreated retinal detachment, pattern dystrophy,achromatopsia, choroideremia, ocular albinism, enhanced S cone syndrome,diabetic retinopathy, retinopathy of prematurity, sickle cellretinopathy, refsun syndrome, Congenital Stationary Night Blindness,glaucoma, gyrate atrophy or retinal vein occlusion. In anotherembodiment, the subject has, or is at risk of developing glaucoma,Leber's hereditary optic neuropathy, lysosomal storage disorder, orperoxisomal disorder. Clinical signs of such ocular diseases include,but are not limited to, decreased peripheral vision, decreased central(reading) vision, decreased night vision, loss of color perception,reduction in visual acuity, decreased photoreceptor function, pigmentarychanges, and ultimately blindness.

In certain embodiments of this invention, the RDH12 nucleic acidsequence, is delivered to the ocular cells in need of treatment by meansof a vector or a viral vector, of which many are known and available inthe art. For delivery to the ocular cells, the therapeutic vector isdesirably non-toxic, non-immunogenic, easy to produce, and efficient inprotecting and delivering DNA into the target cells. A “vector” as usedherein is a nucleic acid molecule into which an exogenous orheterologous or engineered nucleic acid sequence or transgene may beinserted which can then be introduced into an appropriate host cell.Vectors preferably have one or more origin of replication, and one ormore site into which the recombinant DNA can be inserted. Vectors oftenhave convenient means by which cells with vectors can be selected fromthose without, e.g., they encode drug resistance genes. Common vectorsinclude plasmids, viral genomes, and (primarily in yeast and bacteria)“artificial chromosomes.”

The term “exogenous” as used to describe a nucleic acid sequence orprotein means that the nucleic acid or protein does not naturally occurin the position in which it exists in a chromosome, recombinant plasmid,vector or host cell. An exogenous nucleic acid sequence also refers to asequence derived from and inserted into the same host cell or subject,but which is present in a non-natural state, e.g. a different copynumber, or under the control of different regulatory elements.

The term “heterologous” as used to describe a nucleic acid sequence orprotein means that the nucleic acid or protein was derived from adifferent organism or a different species of the same organism than thehost cell or subject in which it is expressed. The term “heterologous”when used with reference to a protein or a nucleic acid in a plasmid,expression cassette, or vector, indicates that the protein or thenucleic acid is present with another sequence or subsequence which withwhich the protein or nucleic acid in question is not found in the samerelationship to each other in nature.

The term “isolated” means that the material is removed from its originalenvironment (e.g., the natural environment if it is naturallyoccurring). For example, a naturally-occurring polynucleotide orpolypeptide present in a living animal is not isolated, but the samepolynucleotide or polypeptide, separated from some or all of thecoexisting materials in the natural system, is isolated, even ifsubsequently reintroduced into the natural system. Such polynucleotidescould be part of a vector and/or such polynucleotides or polypeptidescould be part of a composition, and still be isolated in that suchvector or composition is not part of its natural environment.

By “engineered” is meant that the nucleic acid sequences encoding theRDH12 and RDH12 proteins described herein are assembled and placed intoany suitable genetic element, e.g., naked DNA, phage, transposon,cosmid, episome, etc., which transfers the RDH12 sequences carriedthereon to a host cell, e.g., for generating non-viral delivery systems(e.g., RNA-based systems, naked DNA, or the like) or for generatingviral vectors in a packaging host cell and/or for delivery to a hostcells in a subject. In one embodiment, the genetic element is a plasmid.The methods used to make such engineered constructs are known to thosewith skill in nucleic acid manipulation and include genetic engineering,recombinant engineering, and synthetic techniques. See, e.g., Green andSambrook, Molecular Cloning: A Laboratory Manual, Cold Spring HarborPress, Cold Spring Harbor, N.Y. (2012).

“Virus vectors” are defined as replication defective, synthetic, orrecombinant viral particles containing the exogenous or heterologousnucleic acid sequence encoding the functional RDH12. In one embodiment,an expression cassette as described herein may be engineered onto aplasmid which is used for drug delivery or for production of a viralvector. Suitable viral vectors are preferably replication defective andselected from amongst those which target ocular cells. In oneembodiment, an expression cassette containing a transgene is packaged ina viral capsid or envelope. Any viral genomic sequences also packagedwithin that viral capsid or envelope are replication-deficient; i.e.,they cannot generate progeny virions but retain the ability to infecttarget cells. In one embodiment, the genome of the viral vector does notinclude genes encoding the enzymes required to replicate (the genome canbe engineered to be “gutless”—containing only the transgene of interestflanked by the signals required for amplification and packaging of theartificial genome), but these genes may be supplied during production.Therefore, it is deemed safe for use in gene therapy since replicationand infection by progeny virions cannot occur except in the presence ofthe viral enzyme required for replication.

Viral vectors may include any virus suitable for gene therapy, includingbut not limited to adenovirus; herpes virus; lentivirus; retrovirus;parvovirus, etc. However, for ease of understanding, theadeno-associated virus is referenced herein as an exemplary virusvector. Thus, in one embodiment, a therapeutic composition or reagentcomprises an adeno-associated viral vector comprising an RDH12 transgeneoperatively linked to expression control sequences. The term “transgene”as used herein means an exogenous or engineered protein-encoding nucleicacid sequence that is under the control of a promoter or expressioncontrol sequence in an expression cassette (with or without flankingrAAV ITRs), recombinant plasmid or proviral plasmid, vector, or hostcell described in this specification. In certain embodiments, thetransgene is a codon optimized RDH12 encoding sequence SEQ ID NO: 3. Incertain embodiments, the transgene is a naturally occurring or nativeRDH12 encoding sequence SEQ ID NO:1. In other embodiments, both codonoptimized and natural RDH12 encoding sequences, in various combinationscan serve as the transgene.

As used herein, the term “operably linked” or “operatively associated”refers to both expression control sequences that are contiguous with thenucleic acid sequence encoding the RDH12 and/or expression controlsequences that act in trans or at a distance to control thetranscription and expression thereof

As used herein, the term “host cell” may refer to the packaging cellline in which a recombinant AAV is produced from a proviral plasmid. Inthe alternative, the term “host cell” may refer to any target cell inwhich expression of the transgene is desired. Thus, a “host cell,”refers to a prokaryotic or eukaryotic cell that contains exogenous orheterologous DNA that has been introduced into the cell by any means,e.g., electroporation, calcium phosphate precipitation, microinjection,transformation, viral infection, transfection, liposome delivery,membrane fusion techniques, high velocity DNA-coated pellets, viralinfection and protoplast fusion. In certain embodiments herein, the term“host cell” refers to cultures of ocular cells of various mammalianspecies for in vitro assessment of the compositions described herein. Inother embodiments herein, the term “host cell” refers to the cellsemployed to generate and package the viral vector or recombinant virus.Still in other embodiments, the term “host cell” is intended toreference the ocular cells of the subject being treated in vivo for theocular disease. In yet another embodiment, the host cell can refer toinduced pluripotent stem cells (iPSCs), which are adult cells that havebeen genetically reprogrammed to an embryonic stem cell-like state bybeing forced to express genes and factors important for maintaining thedefining properties of embryonic stem cells, Such cells can bemanipulated and used as models or tools for assessing the function ofthe vectors described herein.

“Plasmids” or plasmid vectors generally are designated herein by a lowercase p preceded and/or followed by capital letters and/or numbers, inaccordance with standard naming conventions that are familiar to thoseof skill in the art. Starting plasmids disclosed herein are eithercommercially available, publicly available on an unrestricted basis, orcan be constructed from available plasmids by routine application ofwell known, published procedures. Many plasmids and other cloning andexpression vectors that can be used in accordance with the presentinvention are well known and readily available to those of skill in theart. Moreover, those of skill readily may construct any number of otherplasmids suitable for use in the invention. The properties, constructionand use of such plasmids, as well as other vectors, in the presentinvention will be readily apparent to those of skill from the presentdisclosure.

As used herein, the term “transcriptional control sequence” or“expression control sequence” refers to nucleic acid sequences, such asinitiator sequences, enhancer sequences, and promoter sequences, whichinduce, repress, or otherwise control the transcription of proteinencoding nucleic acid sequences to which they are operably linked.

In still a further embodiment, a recombinant adeno-associated virus(AAV) vector is provided for delivery of the RDH12 constructs andoptimized sequences described herein. The term “AAV” as used hereinrefers to the dozens of naturally occurring and availableadeno-associated viruses, as well as artificial AAVs. Anadeno-associated virus (AAV) viral vector is an AAV DNase-resistantparticle having an AAV protein capsid into which is packaged nucleicacid sequences for delivery to target cells. An AAV capsid is composedof 60 capsid (cap) protein subunits, VP1, VP2, and VP3, that arearranged in an icosahedral symmetry in a ratio of approximately 1:1:10to 1:1:20, depending upon the selected AAV. AAVs may be selected assources for capsids of AAV viral vectors as identified above. See, e.g.,US Published Patent Application No. 2007-0036760-A1; US Published PatentApplication No. 2009-0197338-A1; EP 1310571. See also, WO 2003/042397(AAV7 and other simian AAV), U.S. Pat. No. 7,790,449 and U.S. Pat. No.7,282,199 (AAV8), WO 2005/033321 and U.S. Pat. No. 7,906,111 (AAV9), andWO 2006/110689, and WO 2003/042397 (rh.10). These documents alsodescribe other AAV which may be selected for generating AAV and areincorporated by reference.

Among the AAVs isolated or engineered from human or non-human primates(NHP) and well characterized, human AAV2 is the first AAV that wasdeveloped as a gene transfer vector; it has been widely used forefficient gene transfer experiments in different target tissues andanimal models. Unless otherwise specified, the AAV capsid, ITRs, andother selected AAV components described herein, may be readily selectedfrom among any AAV, including, without limitation, the AAVs commonlyidentified as AAV1, AAV2, AAV3, AAV4, AAVS, AAV6, AAV7, AAV8, AAV9,AAV8bp, AAV7M8 and AAVAnc80, variants of any of the known or mentionedAAVs or AAVs yet to be discovered or variants or mixtures thereof. See,e.g., WO 2005/033321, which is incorporated herein by reference.

In another embodiment, the AAV capsid is an AAV8bp capsid, whichpreferentially targets bipolar cells. See, WO 2014/024282, which isincorporated herein by reference. In another embodiment, the AAV capsidis an AAV7m8 capsid, which has shown preferential delivery to the outerretina. See, Dalkara et al, In Vivo-Directed Evolution of a NewAdeno-Associated Virus for Therapeutic Outer Retinal Gene Delivery fromthe Vitreous, Sci Transl Med 5, 189ra76 (2013), which is incorporatedherein by reference. In one embodiment, the AAV capsid is an AAV8capsid. In another embodiment, the AAV capsid an AAV9 capsid. In anotherembodiment, the AAV capsid an AAVS capsid.

In some embodiments, an AAV capsid for use in the viral vector can begenerated by mutagenesis (i.e., by insertions, deletions, orsubstitutions) of one of the aforementioned AAV capsids or its encodingnucleic acid. In some embodiments, the AAV capsid is chimeric,comprising domains from two or three or four or more of theaforementioned AAV capsid proteins. In some embodiments, the AAV capsidis a mosaic of Vp1, Vp2, and Vp3 monomers from two or three differentAAVs or recombinant AAVs. In some embodiments, an rAAV compositioncomprises more than one of the aforementioned Caps. In one embodiment,it is desirable to utilize an AAV capsid which shows tropism for thedesired target cell, e.g., photoreceptors, RPE or other ocular cells. Inone embodiment, the AAV capsid is a tyrosine capsid-mutant in whichcertain surface exposed tyrosine residues are substituted withphenylalanine (F).

Such AAV variants are described, e.g., in Mowat et al, January 2014,Tyrosine capsid-mutant AAV vectors for gene delivery to the canineretina from a subretinal or intravitreal approach, Gene Therapy 21,96-105, which is incorporated herein by reference. In one embodiment thecapsid is an AAV8 capsid with a Y733F mutation. In another embodiment,the capsid is an AAV8 capsid with Y447F, Y733F and T494V mutations (alsocalled “AAV8(C&G+T494V)” and “rep2-cap8(Y447F+733F+T494V)”), asdescribed by Kay et al, April 2013, Targeting Photoreceptors viaIntravitreal Delivery Using Novel, Capsid-Mutated AAV Vectors, PLoS One.2013; 8(4): e62097, which is incorporated herein by reference.

As used herein, relating to AAV, the term “variant” means any AAVsequence which is derived from a known AAV sequence, including thosesharing at least 70%, at least 75%, at least 80%, at least 85%, at least90%, at least 95%, at least 97%, at least 99% or greater sequenceidentity over the amino acid or nucleic acid sequence. In anotherembodiment, the AAV capsid includes variants which may include up toabout 10% variation from any described or known AAV capsid sequence.That is, the AAV capsid shares about 90% identity to about 99.9%identity, about 95% to about 99% identity or about 97% to about 98%identity to an AAV capsid provided herein and/or known in the art. Inone embodiment, the AAV capsid shares at least 95% identity with an AAVcapsid. When determining the percent identity of an AAV capsid, thecomparison may be made over any of the variable proteins (e.g., vp1,vp2, or vp3). In one embodiment, the AAV capsid shares at least 95%identity with the AAV8 vp3. In another embodiment, a self-complementaryAAV is used.

The ITRs or other AAV components may be readily isolated or engineeredusing techniques available to those of skill in the art from an AAV.Such AAV may be isolated, engineered, or obtained from academic,commercial, or public sources (e.g., the American Type CultureCollection, Manassas, VA). Alternatively, the AAV sequences may beengineered through synthetic or other suitable means by reference topublished sequences such as are available in the literature or indatabases such as, e.g., GenBank, PubMed, or the like. AAV viruses maybe engineered by conventional molecular biology techniques, making itpossible to optimize these particles for cell specific delivery ofnucleic acid sequences, for minimizing immunogenicity, for tuningstability and particle lifetime, for efficient degradation, for accuratedelivery to the nucleus, etc.

As used herein, “artificial AAV” means, without limitation, an AAV witha non-naturally occurring capsid protein. Such an artificial capsid maybe generated by any suitable technique, using a selected AAV sequence(e.g., a fragment of a vpl capsid protein) in combination withheterologous sequences which may be obtained from a different selectedAAV, non-contiguous portions of the same AAV, from a non-AAV viralsource, or from a non-viral source. An artificial AAV may be, withoutlimitation, a pseudotyped AAV, a chimeric AAV capsid, a recombinant AAVcapsid, or a “humanized” AAV capsid. Pseudotyped vectors, wherein thecapsid of one AAV is replaced with a heterologous capsid protein, areuseful in the invention. In one embodiment, AAV2/5 and AAV2/8 areexemplary pseudotyped vectors.

For packaging an expression cassette or rAAV genome or productionplasmid or proviral plasmid into virions, the ITRs are the only AAVcomponents required in cis in the same construct as the transgene. Inone embodiment, the coding sequences for the replication (rep) and/orcapsid (cap) are removed from the AAV genome and supplied in trans or bya packaging cell line in order to generate the AAV vector. For example,as described above, a pseudotyped AAV may contain ITRs from a sourcewhich differs from the source of the AAV capsid. In one embodiment,AAV2/5 and AAV2/8 are exemplary pseudotyped vectors. Additionally oralternatively, a chimeric AAV capsid may be utilized. Still other AAVcomponents may be selected. Sources of such AAV sequences are describedherein and may also be isolated or engineered obtained from academic,commercial, or public sources (e.g., the American Type CultureCollection, Manassas, VA). Alternatively, the AAV sequences may beobtained through synthetic or other suitable means by reference topublished sequences such as are available in the literature or indatabases such as, e.g., GenBank®, PubMed®, or the like.

“Self-complementary AAV” refers a plasmid or vector having an expressioncassette in which a coding region carried by a recombinant AAV nucleicacid sequence has been designed to form an intra-moleculardouble-stranded DNA template. Upon infection, rather than waiting forcell mediated synthesis of the second strand, the two complementaryhalves of scAAV will associate to form one double stranded DNA (dsDNA)unit that is ready for immediate replication and transcription. See,e.g., D M McCarty et al, “Self-complementary recombinantadeno-associated virus (scAAV) vectors promote efficient transductionindependently of DNA synthesis”, Gene Therapy, (August 2001), Vol 8,Number 16, Pages 1248-1254. Self-complementary AAVs are described in,e.g., U.S. Pat. Nos. 6,596,535; 7,125,717; and 7,456,683, each of whichis incorporated herein by reference in its entirety.

In one embodiment, the AAV is a self-complementary AAV2/8. See, e.g.,Buie et al, January 2010, Self-complementary AAV Virus (scAAV) Safe andLong-term Gene Transfer in the Trabecular Meshwork of Living Rats andMonkeys, Invest Ophthalmol Vis Sci., 51(1): 236-248, and Ryals et al,April 2011, Quantifying transduction efficiencies of unmodified andtyrosine capsid mutant AAV vectors in vitro using two ocular cell lines,Mol Vis.;17:1090-102, which are incorporated herein by reference. In oneembodiment, the AAV is a self-complementary AAV2/8 having at least aY733F mutation. See, Ku et al, Dec. 2011, Gene therapy usingself-complementary Y733F capsid mutant AAV2/8 restores vision in a modelof early onset Leber congenital amaurosis, Hum Mol Genet., 20(23):4569-4581, which is incorporated herein by reference. In anotherembodiment, the AAV is a self-complementary AAV2/8 having at leastY447F+733F+T494V mutations. See, Kay et al, 2013, cited herein.

In one embodiment, the vectors useful in compositions and methodsdescribed herein contain, at a minimum, sequences encoding a selectedAAV capsid, e.g., an AAV8 capsid, or a fragment thereof. In anotherembodiment, useful vectors contain, at a minimum, sequences encoding aselected AAV rep protein, e.g., AAVS rep protein, or a fragment thereof.Optionally, such vectors may contain both AAV cap and rep proteins. Invectors in which both AAV rep and cap are provided, the AAV rep and AAVcap sequences can both be of one origin, e.g., all AAVS origin or allAAV7 origin, etc.

Alternatively, vectors may be used in which the rep sequences are froman AAV which differs from that which is providing the cap sequences. Inone embodiment, the rep and cap sequences are expressed from separatesources (e.g., separate vectors, or a host cell and a vector). Inanother embodiment, these rep sequences are fused in frame to capsequences of a different AAV to form a chimeric AAV vector, such asAAV2/8 described in U.S. Pat. No. 7,282,199, which is incorporated byreference herein.

Certain compositions described herein are isolated, or synthetically orrecombinantly engineered nucleic acid sequences that provide novelcodon-optimized sequences encoding a functional RDH12. In oneembodiment, the optimized nucleic acid sequences encoding the hRDH12constructs described herein are engineered into any suitable geneticelement, e.g., naked DNA, phage, transposon, cosmid, RNA molecule (e.g.,mRNA), episome, etc., which transfers the RDH12 sequences carriedthereon to a host cell, e.g., for generating nanoparticles carrying DNAor RNA, viral vectors in a packaging host cell and/or for delivery to ahost cells in subject. In one embodiment, the genetic element is aplasmid.

The selected genetic element may be delivered by any suitable method,including transfection, electroporation, liposome delivery, membranefusion techniques, high velocity DNA-coated pellets, viral infection andprotoplast fusion. The methods used to make such constructs are known tothose with skill in nucleic acid manipulation and include geneticengineering, recombinant engineering, and synthetic techniques. See,e.g., Green and Sambrook, Molecular Cloning: A Laboratory Manual, ColdSpring Harbor Press, Cold Spring Harbor, N.Y. (2012).

A variety of expression cassettes are provided which employ SEQ ID NO. 3for expression of multiple or different versions of the hRDH12 protein.As used herein, an “expression cassette” refers to a nucleic acidmolecule which comprises coding sequences for the optimized RDH12proteins, promoter, and may include other regulatory sequences therefor,which cassette may be engineered into a genetic element or plasmid,and/or packaged into the capsid of a viral vector (e.g., a viralparticle). In one embodiment, an expression cassette comprises a codonoptimized nucleic acid sequence that encodes RDH12. In one embodiment,the cassette provides the codon optimized RDH12 operatively associatedwith expression control sequences that direct expression of the codonoptimized nucleic acid sequence that encodes RDH12 in a host cell.

In another embodiment, an expression cassette comprises a codonoptimized nucleic acid sequence that encodes RDH12. In one embodiment,the cassette provides the codon optimized RDH12 operatively associatedwith expression control sequences that direct expression of the codonoptimized nucleic acid sequence that encodes RDH12 in a host cell.

In still another embodiment, an expression cassette comprises a codonoptimized nucleic acid sequence that encodes a functional, optimizedRDH12. In one embodiment of such an expression cassette, the sequenceencoding RDH12 is operatively associated with the a first expressioncontrol sequence(s) that direct expression of a naturally occurringnucleic acid sequence that encodes RDH12 in a host cell. As describedabove for the expression cassettes containing RDH12, in embodimentswhich express mulitple copies or multiple different versions offunctional RDH12, the codon optimized sequence may be positioned, 5′ or3′ to another version of the sequences. One of skill in the art mayreadily design constructs for expression of multiple copies of RDH12 inview of the teachings of this specification and the prior art.

As described herein, an expression cassette can be flanked on its 5′ endby a 5′AAV inverted terminal repeat sequence (ITR) and on its 3′ end bya 3′ AAV ITR. Thus, this rAAV ITR-flanked expression cassette containsthe minimal sequences required to package the expression cassette intoan AAV viral particle, i.e., the AAV 5′ and 3′ ITRs. The AAV ITRs may beobtained from the ITR sequences of any AAV, such as described herein.These ITRs may be of the same AAV origin as the capsid employed in theresulting recombinant AAV, or of a different AAV origin (to produce anAAV pseudotype). In one embodiment, the ITR sequences from AAV2, or thedeleted version thereof (ΔITR), are used for convenience and toaccelerate regulatory approval. However, ITRs from other AAV sources maybe selected. Each rAAV genome can be then introduced into a proviralplasmid following the teachings of WO2012/158757. The proviral plasmidsare cultured in the host cells which express the AAV cap and/or repproteins. In the host cells, each rAAV genome is rescued and packagedinto the capsid protein or envelope protein to form an infectious viralparticle.

In yet another embodiment, a vector comprising any of the expressioncassettes described herein is provided. As described above, such vectorscan be plasmids of variety of origins and are useful in certainembodiments for the generation of recombinant replication defectiveviruses as described further herein.

In one another embodiment, the vector is a proviral plasmid thatcomprises an AAV capsid and an recombinant AAV-ITR flanked expressioncassette, wherein said cassette comprises a codon optimized nucleic acidsequence that encodes RDH12 or multiple (i.e., at least two) copies ofRDH12, and expression control sequences that direct expression of theencoded protein in a host cell.

One type of proviral plasmid comprises a modular recombinant expressioncassette that permits portions of the components of cassette to beremoved and repeatedly replaced with other components without destroyingthe restriction sites in the plasmid. Such a proviral plasmid is onethat contains a 5′ AAV ITR sequence, the ITR flanked upstream byrestriction site 1 and downstream by restriction site 2; a selectedpromoter flanked upstream by restriction site 2 and downstream byrestriction site 3. Another component of the modular rAAV is apolylinker sequence comprising at least restriction site 3, restrictionsite 4 and restriction site 5, that contains a codon optimized nucleicacid sequence that encodes RDH12, or two or more copies of a sequencethat encodes RDH12, at least one such sequence being a codon optimizednucleic acid sequence encoding RDH12. The RDH12 encoding sequences arelocated between any two of the restriction sites 3, 4 and 5, and areoperatively linked to, and under the regulatory control of, thepromoter. Alternatively, the second encoding sequence is inserted intothe polylinker sequence along with the second expression controlsequence of the expression cassette as described above.

Additional components of the modular rAAV cassette include apolyadenylation sequence flanked upstream by restriction site 4 or 5 anddownstream by restriction site 6;

and a 3′ AAV ITR sequence flanked upstream by restriction site 6 anddownstream by restriction site 7. The proviral plasmid also containselements necessary for replication in bacterial cells, and a resistancegene. Each of the above-noted restriction sites 1 through 7 occurs onlyonce in the proviral plasmid and is cleaved by a different enzyme thatcannot cleave another restriction site in the plasmid and thereby permitindependent and repeated removal, replacement or substitution of theentire rAAV modular cassette or only the elements flanked by thoserestriction sites from the plasmid. Such plasmids are described indetail in International Patent Application Publication No.WO2012/158757, incorporated by reference herein.

A suitable recombinant adeno-associated virus (AAV) is generated byculturing a host cell which contains a nucleic acid sequence encoding anadeno-associated virus (AAV) serotype capsid protein, or fragmentthereof, as defined herein; a functional rep gene; a minigene composedof, at a minimum, AAV inverted terminal repeats (ITRs) and a NPHPSnucleic acid sequence; and sufficient helper functions to permitpackaging of the minigene into the AAV capsid protein. The componentsrequired to be cultured in the host cell to package an AAV minigene inan AAV capsid may be provided to the host cell in trans. Alternatively,any one or more of the required components (e.g., minigene, repsequences, cap sequences, and/or helper functions) may be provided by astable host cell which has been engineered to contain one or more of therequired components using methods known to those of skill in the art.

Most suitably, such a stable host cell will contain the requiredcomponent(s) under the control of an inducible promoter. However, therequired component(s) may be under the control of a constitutivepromoter. Examples of suitable inducible and constitutive promoters areprovided herein, in the discussion below of regulatory elements suitablefor use with the transgene, i.e., RDH12. In still another alternative, aselected stable host cell may contain selected component(s) under thecontrol of a constitutive promoter and other selected component(s) underthe control of one or more inducible promoters. For example, a stablehost cell may be generated which is derived from 293 cells (whichcontain El helper functions under the control of a constitutivepromoter), but which contains the rep and/or cap proteins under thecontrol of inducible promoters. Still other stable host cells may begenerated by one of skill in the art.

The minigene, rep sequences, cap sequences, and helper functionsrequired for producing the rAAV of the invention may be delivered to thepackaging host cell in the form of any genetic element which transfersthe sequences carried thereon. The selected genetic element may bedelivered by any suitable method, including those described herein. Themethods used to construct any embodiment of this invention are known tothose with skill in nucleic acid manipulation and include geneticengineering, recombinant engineering, and synthetic techniques. See,e.g., Sambrook et al, Molecular Cloning: A Laboratory Manual, ColdSpring Harbor Press, Cold Spring Harbor, N.Y. Similarly, methods ofgenerating rAAV virions are well known and the selection of a suitablemethod is not a limitation on the present invention. See, e.g., K.Fisher et al, 1993 1 Virol., 70:520-532 and U.S. Pat. No. 5,478,745,among others. These publications are incorporated by reference herein.

The minigene is composed of, at a minimum, a RDH12 nucleic acid sequence(the transgene), as described above, and its regulatory sequences, and5′ and 3′ AAV inverted terminal repeats (ITRs). In one desirableembodiment, the ITRs of AAV serotype 2 are used. However, ITRs fromother suitable serotypes may be selected. It is this minigene which ispackaged into a capsid protein and delivered to a selected host cell.

The regulatory sequences include conventional control elements which areoperably linked to the RDH12 gene in a manner which permits itstranscription, translation and/or expression in a cell transfected withthe vector or infected with the virus produced by the invention. As usedherein, “operably linked” sequences include both expression controlsequences that are contiguous with the gene of interest and expressioncontrol sequences that act in trans or at a distance to control the geneof interest.

Expression control sequences include appropriate transcriptioninitiation, termination, promoter and enhancer sequences; efficient RNAprocessing signals such as splicing and polyadenylation (polyA) signals;sequences that stabilize cytoplasmic mRNA; sequences that enhancetranslation efficiency (i.e., Kozak consensus sequence); sequences thatenhance protein stability; and when desired, sequences that enhancesecretion of the encoded product. A great number of expression controlsequences, including promoters, are known in the art and may beutilized.

The regulatory sequences useful in the constructs of the presentinvention may also contain an intron, desirably located between thepromoter/ enhancer sequence and the gene. One desirable intron sequenceis derived from SV-40, and is a 100 bp mini-intron splice donor/spliceacceptor referred to as SD-SA. Another suitable sequence includes thewoodchuck hepatitis virus post-transcriptional element. (See, e.g., L.Wang and I. Verma, 1999 Proc. Natl. Acad. Sci., USA, 96:3906-3910).PolyA signals may be derived from many suitable species, including,without limitation SV-40, human and bovine.

Another regulatory component of the rAAV useful in the method of theinvention is an internal ribosome entry site (IRES). An IRES sequence,or other suitable system, may be used to produce more than onepolypeptide from a single gene transcript. An IRES (or other suitablesequence) is used to produce a protein that contains more than onepolypeptide chain or to express two different proteins from or withinthe same cell. An exemplary IRES is the poliovirus internal ribosomeentry sequence, which supports transgene expression in photoreceptors,RPE and ganglion cells. Preferably, the IRES is located 3′ to thetransgene in the rAAV vector.

The selection of the promoter to be employed in the rAAV may be madefrom among a wide number of constitutive or inducible promoters that canexpress the selected transgene in the desired an ocular cell. In anotherembodiment, the promoter is cell-specific. The term “cell-specific”means that the particular promoter selected for the recombinant vectorcan direct expression of the selected transgene in a particular ocularcell type. In one embodiment, the promoter is specific for expression ofthe transgene in photoreceptor cells. In another embodiment, thepromoter is specific for expression in the rods and cones. In anotherembodiment, the promoter is specific for expression in the rods. Inanother embodiment, the promoter is specific for expression in thecones. In another embodiment, the promoter is specific for expression ofthe transgene in RPE cells. In another embodiment, the transgene isexpressed in any of the above noted ocular cells.

The promoter may be derived from any species. Exemplary promoters may bethe human G-protein-coupled receptor protein kinase 1 (GRK1) promoter(Genbank Accession number AY327580). In another embodiment, the promoteris a 292 nt fragment (positions 1793-2087) of the GRK1 promoter (See,Beltran et al, Gene Therapy 2010 17:1162-74, which is herebyincorporated by reference herein). In another preferred embodiment, thepromoter is the human interphotoreceptor retinoid-binding proteinproximal (IRBP) promoter. In one embodiment, the promoter is a 235 ntfragment of the hIRBP promoter.

In another embodiment, promoter is the native promoter for the gene tobe expressed. In one embodiment, the promoter is the RDH12 proximalpromoter. Other promoters useful in the invention include, withoutlimitation, the RPGR proximal promoter (Shu et al, IOVS, May 2102), therod opsin promoter, the red-green opsin promoter, the blue opsinpromoter, the cGMP-β-phosphodiesterase promoter, the mouse opsinpromoter (Beltran et al 2010 cited above), the rhodopsin promoter(Mussolino et al, Gene Ther, July 2011, 18(7):637-45); the alpha-subunitof cone transducin (Morrissey et al, BMC Dev, Biol, January 2011, 11:3);beta phosphodiesterase (PDE) promoter; the retinitis pigmentosa (RP1)promoter (Nicord et al, J. Gene Med, Dec 2007, 9(12):1015-23); theNXNL2/NXNL1 promoter (Lambard et al, PLoS One, Oct. 2010, 5(10):e13025),the RPE65 promoter; the retinal degeneration slow/peripherin 2(Rds/perph2) promoter (Cai et al, Exp Eye Res. 2010 August;91(2):186-94); and the VMD2 promoter (Kachi et al, Human Gene Therapy,2009 (20:31-9)). Each of these documents is incorporated by referenceherein. In another embodiment, the promoter is selected from human EF1αpromoter, rhodopsin promoter, rhodopsin kinase, interphotoreceptorbinding protein (IRBP), cone opsin promoters (red-green, blue), coneopsin upstream sequences containing the red-green cone locus controlregion, cone transducing, and transcription factor promoters (neuralretina leucine zipper (Nr1) and photoreceptor-specific nuclear receptorNr2e3, bZIP).

In one embodiment, the promoter is of a small size, under 1000 bp, dueto the size limitations of the AAV vector. In another embodiment, thepromoter is under 400 bp. Other promoters may be selected by one ofskill in the art.

In another embodiment, the promoter is a ubiquitous or constitutivepromoter. An example of a suitable promoter is a hybrid chicken (β-actin(CBA) promoter with cytomegalovirus (CMV) enhancer elements. In anotherembodiment, the promoter is the CB7 promoter. Other suitable promotersinclude the human β-actin promoter, the human elongation factor-lapromoter, the cytomegalovirus (CMV) promoter, the simian virus 40promoter, and the herpes simplex virus thymidine kinase promoter. See,e.g., Damdindorj et al, (August 2014) A Comparative Analysis ofConstitutive Promoters Located in Adeno-Associated Viral Vectors. PLoSONE 9(8): e106472. Still other suitable promoters include viralpromoters, constitutive promoters, regulatable promoters [see, e.g., WO2011/126808 and WO 2013/04943]. Alternatively a promoter responsive tophysiologic cues may be utilized in the expression cassette, rAAVgenomes, vectors, plasmids and viruses described herein. In oneembodiment, the promoter is of a small size, under 1000 bp, due to thesize limitations of the AAV vector. In another embodiment, the promoteris under 400 bp. Other promoters may be selected by one of skill in theart.

Examples of constitutive promoters useful in the invention include,without limitation, the retroviral Rous sarcoma virus (RSV) LTR promoter(optionally with the RSV enhancer), the cytomegalovirus (CMV) promoter(optionally with the CMV enhancer), the SV40 promoter, the dihydrofolatereductase promoter, the chicken β-actin (CBA) promoter, thephosphoglycerol kinase (PGK) promoter, the EF1 promoter (Invitrogen),and the immediate early CMV enhancer coupled with the CBA promoter(Beltran et al, Gene Therapy 2010 cited above).

Inducible promoters allow regulation of gene expression and can beregulated by exogenously supplied compounds, environmental factors suchas temperature, or the presence of a specific physiological state, e.g.,acute phase, a particular differentiation state of the cell, or inreplicating cells only. Inducible promoters and inducible systems areavailable from a variety of commercial sources, including, withoutlimitation, Invitrogen, Clontech and Ariad. Many other systems have beendescribed and can be readily selected by one of skill in the art.Examples of inducible promoters regulated by exogenously suppliedcompounds, include, the zinc-inducible sheep metallothionine (MT)promoter, the dexamethasone (Dex)-inducible mouse mammary tumor virus(MMTV) promoter, the T7 polymerase promoter system; the ecdysone insectpromoter, the tetracycline-repressible system, thetetracycline-inducible system, the RU486-inducible system and therapamycin-inducible system. Other types of inducible promoters which maybe useful in this context are those which are regulated by a specificphysiological state, e.g., temperature, acute phase, a particulardifferentiation state of the cell, or in replicating cells only. Anytype of inducible promoter which is tightly regulated and is specificfor the particular target ocular cell type may be used.

In other embodiments, the cassette, vector, plasmid and virus constructsdescribed herein contain other appropriate transcription initiation,termination, enhancer sequences, efficient RNA processing signals suchas splicing and polyadenylation (polyA) signals; TATA sequences;sequences that stabilize cytoplasmic mRNA; sequences that enhancetranslation efficiency (i.e., Kozak consensus sequence); introns;sequences that enhance protein stability; and when desired, sequencesthat enhance secretion of the encoded product. The expression cassetteor vector may contain none, one or more of any of the elements describedherein. Examples of suitable polyA sequences include, e.g., SV40, bovinegrowth hormone (bGH), and TK polyA. Examples of suitable enhancersinclude, e.g., the CMV enhancer, the RSV enhancer, the alpha fetoproteinenhancer, the TTR minimal promoter/enhancer, LSP (TH-binding globulinpromoter/alpha1-microglobulin/bikunin enhancer), amongst others.

Other enhancer sequences useful in the invention include the IRBPenhancer (Nicord 2007, cited above), immediate early cytomegalovirusenhancer, one derived from an immunoglobulin gene or SV40 enhancer, thecis-acting element identified in the mouse proximal promoter, etc.

Selection of these and other common vector and regulatory elements areconventional and many such sequences are available. See, e.g., Sambrooket al, and references cited therein at, for example, pages 3.18-3.26 and16.17-16.27 and Ausubel et al., Current Protocols in Molecular Biology,John Wiley & Sons, New York, 1989). Of course, not all vectors andexpression control sequences will function equally well to express allof the transgenes of this invention. However, one of skill in the artmay make a selection among these, and other, expression controlsequences without departing from the scope of this invention.

Methods for generating and isolating AAV viral vectors suitable fordelivery to a subject are known in the art. See, e.g., U.S. Pat. Nos.7,790,449; 7,282,199; WO 2003/042397; WO 2005/033321, WO 2006/110689;and U.S. Pat. No. 7,588,772 B2]. In a one system, a producer cell lineis transiently transfected with a construct that encodes the transgeneflanked by ITRs and a construct(s) that encodes rep and cap. In a secondsystem, a packaging cell line that stably supplies rep and cap istransiently transfected with a construct encoding the transgene flankedby ITRs. In each of these systems, AAV virions are produced in responseto infection with helper adenovirus or herpesvirus, requiring theseparation of the rAAVs from contaminating virus. More recently, systemshave been developed that do not require infection with helper virus torecover the AAV - the required helper functions (i.e., adenovirus E1,E2a, VA, and E4 or herpesvirus UL5, UL8, UL52, and UL29, and herpesviruspolymerase) are also supplied, in trans, by the system. In these newersystems, the helper functions can be supplied by transient transfectionof the cells with constructs that encode the required helper functions,or the cells can be engineered to stably contain genes encoding thehelper functions, the expression of which can be controlled at thetranscriptional or posttranscriptional level.

In yet another system, the transgene flanked by ITRs and rep/cap genesare introduced into insect cells by infection with baculovirus-basedvectors. For reviews on these production systems, see generally, e.g.,Zhang et al., 2009, “Adenovirus-adeno-associated virus hybrid forlarge-scale recombinant adeno-associated virus production,” Human GeneTherapy 20:922-929, the contents of each of which is incorporated hereinby reference in its entirety. Methods of making and using these andother AAV production systems are also described in the following, thecontents of each of which is incorporated herein by reference in itsentirety: U.S. Pat. Nos. 5,139,941; 5,741,683; 6,057,152; 6,204,059;6,268,213; 6,491,907; 6,660,514; 6,951,753; 7,094,604; 7,172,893;7,201,898; 7,229,823; and 7,439,065. See generally, e.g., Grieger &Samulski, 2005, “Adeno-associated virus as a gene therapy vector: Vectordevelopment, production and clinical applications,” Adv. Biochem.Engin/Biotechnol. 99: 119-145; Buning et al., 2008, “Recent developmentsin adeno-associated virus vector technology,” J. Gene Med. 10:717-733;and the references cited below, each of which is incorporated herein byreference in its entirety.

The methods used to construct any embodiment of this invention are knownto those with skill in nucleic acid manipulation and include geneticengineering, recombinant engineering, and synthetic techniques. See,e.g., Green and Sambrook et al, Molecular Cloning: A Laboratory Manual,Cold Spring Harbor Press, Cold Spring Harbor, N.Y. (2012). Similarly,methods of generating rAAV virions are well known and the selection of asuitable method is not a limitation on the present invention. See, e.g.,K. Fisher et al, (1993) J. Virol., 70:520-532 and U.S. Pat. No.5,478,745.

In other embodiments, the cassette, vector, plasmid and virus constructsdescribed herein contain other appropriate transcription initiation,termination, enhancer sequences, efficient RNA processing signals suchas splicing and polyadenylation (polyA) signals; TATA sequences;sequences that stabilize cytoplasmic mRNA; sequences that enhancetranslation efficiency (i.e., Kozak consensus sequence); introns;sequences that enhance protein stability; and when desired, sequencesthat enhance secretion of the encoded product. The expression cassetteor vector may contain none, one or more of any of the elements describedherein. Examples of suitable polyA sequences include, e.g., SV40, bovinegrowth hormone (bGH), and TK polyA. Examples of suitable enhancersinclude, e.g., the CMV enhancer, the RSV enhancer, the alpha fetoproteinenhancer, the TTR minimal promoter/enhancer, LSP (TH-binding globulinpromoter/alpha1-microglobulin/bikunin enhancer), amongst others.

Thus, in one embodiment novel AAV proviral plasmids carrying native oroptimized cDNAs of Retinal Dehydrogenase 12 (RDH12) that encode normalor functional RDH12 protein, such plasmids are capable of being packagedin the AAV vectors. These vectors have demonstrated biologicalactivities both in vitro and in vivo.

In one embodiment, a method of generating a recombinant rAAV comprisesobtaining a plasmid containing a rAAV genome as described above andculturing a packaging cell carrying the plasmid in the presence ofsufficient viral sequences to permit packaging of the AAV viral genomeinto an infectious AAV envelope or capsid. Specific methods of rAAVvector generation are described above and may be employed in generatinga rAAV vector that can deliver one or more of the codon optimized RDH12or RDH12 in the expression cassettes and genomes described above and inthe examples below.

In still another aspect, a method of gene editing is employed to correcta mutation or undesirable RDH12 gene sequence in the eye. One desirablemethod of gene editing employs the “CRISPR/Cas9” system. CRISPR/Cas is atechnique which has been described as having potential in correction ofdiseases associated with a genetic mutation or a specific phenotype. TheClustered Regularly Interspaced Short Palindromic Repeats (CRISPR) andCRISPR-associated protein (Cas9) system has two distinct components: (1)a guide RNA and (2) an endonuclease, in this case the CRISPR associated(Cas) nuclease, Cas9. The guide RNA is a combination of endogenousbacterial crRNA (CRISPR RNA) and tracrRNA (transactivating crRNA) into asingle chimeric guide RNA (gRNA) transcript. The gRNA combines thetargeting specificity of crRNA with the scaffolding properties oftracrRNA into a single transcript. When the gRNA and the Cas9 areexpressed in the cell, the genomic target sequence can be modified orpermanently disrupted. This system has been employed for genomicengineering for mammalian systems, see, e.g., Cong, L., et al. 2013.Science 339: 819-823; Mali, P., et al. 2013 Science 339: 823-826; Ran,F.A., et al. 2013. Nat. Protoc. 8: 2281-2308; and Shalem, 0., et al.2014 Science 343: 84-87. The CRISPR Type II system is currently the mostcommonly used RNA-guided endonuclease technology for genome engineering.Adeno-associated viruses have been described as being useful vectors forgene therapy involving the CRISPR-Cas system. See, e.g., Yin et al, 2014Biotechnology, 32: 551-3 and 2015 Nature Biotechnology, 33: 102-6.

In still another embodiment, these gene editing techniques can beemployed on selected cells to insert the RDH12-encoding sequence (nativeor codon-optimized). Such cells can include HEPG2 cells or inducedpluripotent stem cells (iPSCs), which can then be used as models toexamine the functions of the vectors, recombinant viruses, and othercompositions and reagents herein.

In yet other aspects, these nucleic acid sequences, vectors, proviralplasmids, expression cassettes, rAAV-ITR flanked expression cassettes,viral vectors, including rAAV viral vectors are useful in pharmaceuticalcompositions, which also comprise a pharmaceutically acceptable carrier.Such pharmaceutical compositions are used to express the optimized RDH12or multiple copies of RDH12 in the ocular cells through delivery by suchrecombinantly engineered AAVs or artificial AAV's.

To prepare these ophthalmic pharmaceutical compositions containing thenucleic acid sequences, vectors, rAAV genomes and rAAV viral vectors,the sequences or vectors or viral vector are preferably assessed forcontamination by conventional methods and then formulated into apharmaceutical composition suitable for administration to the eye. Ingeneral, ophthalmic pharmaceutical preparations are sterile formulationsessentially free from foreign particles, suitably compounded andpackaged for instillation in to the eye. In one embodiment, theformulation is suitable for subretinal injection. Such formulationinvolves the use of a pharmaceutically and/or physiologically acceptablevehicle or carrier, particularly one suitable for administration to theeye. Suitable pharmaceutical carriers including phosphate bufferedsaline solutions, water, emulsions, such as oil/water emulsions ormicroemulsions, suspensions, various types of wetting agents, sterilesolutions, in liposomes, in niosomes, discomes, or even in ointment orgels., such as buffered saline or other buffers, e.g., HEPES, tomaintain pH at appropriate physiological levels.

Optionally, other medicinal agents, such as pharmaceutical agents,stabilizing agents, buffers, adjuvants, diluents, or surfactants, etc.are included. For injection, the carrier will typically be a liquid.Exemplary physiologically acceptable carriers include sterile,pyrogen-free water and sterile, pyrogen-free, phosphate buffered saline.A variety of such known carriers are provided in US Patent PublicationNo. 7,629,322, incorporated herein by reference. In one embodiment, thecarrier is an isotonic sodium chloride solution. In another embodiment,the carrier is balanced salt solution. In one embodiment, the carrierincludes Tween. If the virus is to be stored long-term, it may be frozenin the presence of glycerol or Tween20.

In one embodiment, the contents of the formulation are designed tomaintain a pH range of 4.75 to 7.40. Topical products also requireadjustment of tonicity close to that of natural tears. Generally, arange of 0.5% to 2% saline tonicity is well-tolerated. Suitablesurfactants also include, for example, synthetic surfactants, such ascolfosceril palmitate (Exosurf), a mixture of DPPC with hexadecanol andtyloxapol added as spreading agents; Pumactant, a mixture of DPPC andPG; KL-4 which is composed of DPPC, palmitoyl-oleoylphosphatidylglycerol, and palmitic acid, combined with a 21 amino acidsynthetic peptide that mimics the structural characteristics of SP-B;Venticute, a combination of DPPC, PG, palmitic acid and recombinantSP-C; or animal derived surfactants such as Beractant (Alveofact orSurvanta), Calfactant (Infasurf) or Poractant alfa (Curosurf). Anotheruseful surfactant is Surfaxin (an FDA approved synthetic peptide. Stillanother useful surfactant is Pluronic F68.

In one exemplary specific embodiment, a suitable formulation contains180 mM NaCl, 10 mM sodium phosphate buffer (NaPi), pH 7.3 with 0.0001%-0.01% Pluronic F68 (PF68) surfactant. The exact composition of thesaline component of the buffer ranges from 160 mM to 180 mM NaCl.Optionally a different pH buffer (potentially HEPEs, sodium bicarbonate,or TRIS) is used in place of the buffer specifically described. Stillalternatively, a buffer containing 0.9% NaCl is useful.

Optionally, the compositions of the invention may contain, in additionto the rAAV and/or variants and carrier(s), other conventionalpharmaceutical ingredients, such as preservatives, or chemicalstabilizers. Suitable exemplary preservatives include chlorobutanol,potassium sorbate, sorbic acid, sulfur dioxide, propyl gallate, theparabens, ethyl vanillin, glycerin, phenol, and parachlorophenol.Suitable chemical stabilizers include gelatin and albumin.

The pharmaceutical compositions containing a replication-defective rAAVviruses can be formulated with a physiologically acceptable carrier foruse in gene transfer and gene therapy applications. In the case of AAVviral vectors, quantification of the genome copies (“GC”), vectorgenomes, or virus particles may be used as the measure of the dosecontained in the formulation or suspension. Any method known in the artcan be used to determine the genome copy (GC) number of thereplication-defective virus compositions of the invention. One methodfor performing AAV GC number titration is as follows: Purified AAVvector samples are first treated with DNase to eliminate un-encapsidatedAAV genome DNA or contaminating plasmid DNA from the production process.The DNase resistant particles are then subjected to heat treatment torelease the genome from the capsid. The released genomes are thenquantitated by real-time PCR using primer/probe sets targeting specificregion of the viral genome (usually poly A signal). In another methodthe effective dose of a recombinant adeno-associated virus carrying anucleic acid sequence encoding the optimized RDH12 transgene under thedesirably are measured as described in S. K. McLaughlin et al, 1988 J.Virol., 62:1963. In another method, the titer is determined usingdroplet digital PCR (ddPCR). See, Lock as described in, e.g., M. Lock etal, Hu Gene Therapy Methods, 2014 April; 25(2):115-25. doi:10.1089/hgtb.2013.131. Epub 2014 Feb. 14, which is incorporated hereinby reference.

By “administering” as used in the methods means delivering thecomposition to the target selected cell which is characterized by theocular disease. In one embodiment, the method involves delivering thecomposition by subretinal injection to the photoreceptor cells or otherocular cells. In another embodiment, intravitreal injection to ocularcells is employed. In still another method, injection via the palpebralvein to ocular cells may be employed. Still other methods ofadministration may be selected by one of skill in the art given thisdisclosure. By “administering” or “route of administration” is deliveryof composition described herein, with or without a pharmaceuticalcarrier or excipient, of the subject. Routes of administration may becombined, if desired. In some embodiments, the administration isrepeated periodically. The pharmaceutical compositions described hereinare designed for delivery to subjects in need thereof by any suitableroute or a combination of different routes. Direct delivery to the eye(optionally via ocular delivery, intra-retinal injection, intravitreal,topical), or delivery via systemic routes, e.g., intraarterial,intraocular, intravenous, intramuscular, subcutaneous, intradermal, andother parental routes of administration. The nucleic acid moleculesand/or vectors described herein may be delivered in a single compositionor multiple compositions. Optionally, two or more different AAV may bedelivered, or multiple viruses [see, e.g., WO20 2011/126808 and WO2013/049493]. In another embodiment, multiple viruses may containdifferent replication-defective viruses (e.g., AAV and adenovirus),alone or in combination with proteins.

As used herein, the term “treatment” or “treating” is definedencompassing administering to a subject one or more compounds orcompositions described herein for the purposes of amelioration of one ormore symptoms of an ocular disease. “Treatment” can thus include one ormore of reducing onset or progression of an ocular disease, preventingdisease, reducing the severity of the disease symptoms, or retardingtheir progression, including the progression of blindness, removing thedisease symptoms, delaying onset of disease or monitoring progression ofdisease or efficacy of therapy in a given subject.

As used herein, the term “dosage” can refer to the total dosagedelivered to the subject in the course of treatment, or the amountdelivered in a single unit (or multiple unit or split dosage)administration. The pharmaceutical virus compositions can be formulatedin dosage units to contain an amount of replication-defective viruscarrying the nucleic acid sequences encoding RDH12 as described herein.Dosages can be expressed in genome copies (GC) of the nucleic acidsequences. Dosages can also be expressed in terms of viral particles. Inone embodiment, a suitable dosage is in the range of about 1.0×10⁶ GC orviral particles to about 1.0×10¹⁵ GC including all integers orfractional amounts within the range. In one embodiment, the compositionsare formulated to contain, or dosages administered in amounts of, atleast 1×10⁶, 2×10⁶, 3×10⁶, 4×10⁶, 5×10⁶, 6×10⁶, 7×10⁶, 8×10⁶, or 9×10⁶GC per dose including all integers or fractional amounts within therange. In one embodiment, the compositions are formulated to contain, ordosages administered in amounts of, at least 1×10⁷, 2×10⁷, 3×10⁷, 4×10⁷,5×10⁷, 6×10⁷, 7×10⁷, 8×10⁷, or 9×10⁷ GC per dose including all integersor fractional amounts within the range. In one embodiment, thecompositions are formulated to contain at least 1×10⁸, 2×10⁸, 3×10⁸,4×10⁸, 5×10⁸, 6×10⁸, 7×10⁸, 8×10⁸, or 9×10⁸ GC per dose including allintegers or fractional amounts within the range. In one embodiment, thecompositions are formulated to contain at least 1×10⁹, 2×10⁹, 3×10⁹,4×10⁹, 5×10⁹, 6×10⁹, 7×10⁹, 8×10⁹, or 9×10⁹ GC per dose including allintegers or fractional amounts within the range. In another embodiment,the compositions are formulated to contain at least 1×10¹¹, 2×10¹¹,3×10¹⁰, 4×10¹⁰, 5×10¹⁰, 6×10¹⁰, 7×10¹⁰, 8×10¹⁰, or 9×10¹⁰ GC per doseincluding all integers or fractional amounts within the range. Inanother embodiment, the compositions are formulated to contain at least1×10¹¹, 2×10¹¹, 3×10¹¹, 4×10¹¹, 5×10¹¹, 6×10¹¹, 7×10¹¹, 8×10¹¹, or9×10¹¹ GC per dose including all integers or fractional amounts withinthe range. In another embodiment, the compositions are formulated tocontain at least 1×10¹², 2×10¹², 3×10¹², 4×10¹², 5×10¹², 6×10¹², 7×10¹²,8×10¹², or 9×10¹² GC per dose including all integers or fractionalamounts within the range. In another embodiment, the compositions areformulated to contain at least 1×10¹³, 2×10¹³, 3×10¹³, 4×10¹³, 5×10¹³,6×10¹³, 7×10¹³, 8×10¹³, or 9×10¹³ GC per dose including all integers orfractional amounts within the range. In another embodiment, thecompositions are formulated to contain at least 1×10¹⁴, 2×10¹⁴, 3×10¹⁴,4×10¹⁴, 5×10¹⁴, 6×10¹⁴, 7×10¹⁴, 8×10¹⁴, or 9×10¹⁴ GC per dose includingall integers or fractional amounts within the range. In anotherembodiment, the compositions are formulated to contain at least 1×10¹⁵,2×10¹⁵, 3×10¹⁵, 4×10¹⁵, 5×10¹⁵, 6×10¹⁵, 7×10¹⁵, 8×10¹⁵, or 9×10¹⁵ GC perdose including all integers or fractional amounts within the range. Inone embodiment, for human application the dose can range from 1×10⁶toabout 1×10¹³ GC per dose including all integers or fractional amountswithin the range. In one embodiment, for human application the dose canrange from 1×10¹⁰ to about 1×10¹² GC per dose including all integers orfractional amounts within the range. Still other doses and dosages maybe determined by the attending physician.

These above doses may be administered in a variety of volumes ofcarrier, excipient or buffer formulation, ranging from about 25 to about1000 microliters, including all numbers within the range, depending onthe size of the area to be treated, the viral titer used, the route ofadministration, and the desired effect of the method. In one embodiment,the volume of carrier, excipient or buffer is at least about 25 μl. Inone embodiment, the volume is about 50 μl. A In another embodiment, thevolume is about 75 μl. In another embodiment, the volume is about 100μl. In another embodiment, the volume is about 125 μl. In anotherembodiment, the volume is about 150 μl. In another embodiment, thevolume is about 175 μl. In yet another embodiment, the volume is about200 μL. In another embodiment, the volume is about 225 μl. In yetanother embodiment, the volume is about 250 μl. In yet anotherembodiment, the volume is about 275 μl. In yet another embodiment, thevolume is about 300 μL. In yet another embodiment, the volume is about325 μL. In another embodiment, the volume is about 350 μl. In anotherembodiment, the volume is about 375 μl. In another embodiment, thevolume is about 400 μl. In another embodiment, the volume is about 450μl. n another embodiment, the volume is about 500 μl. In anotherembodiment, the volume is about 550 μl. In another embodiment, thevolume is about 600 μl. In another embodiment, the volume is about 650μl. In another embodiment, the volume is about 700 μl. In anotherembodiment, the volume is between about 700 and 1000 μl.

In one embodiment, the viral constructs may be delivered inconcentrations of from at least least 1×10⁶ to about least 1×10¹¹ GCs involumes of about 1 μl to about 3 μl for small animal subjects, such asmice. For larger veterinary subjects having eyes about the same size ashuman eyes, the larger human dosages and volumes stated above, e.g.,1×10⁶ to about 1×10¹⁵ GC per dose, are useful. See, e.g., Diehl et al,J. Applied Toxicology, 21:15-23 (2001) for a discussion of goodpractices for administration of substances to various veterinaryanimals. This document is incorporated herein by reference.

It is desirable that the lowest effective concentration of virus orother delivery vehicle be utilized in order to reduce the risk ofundesirable effects, such as toxicity, retinal dysplasia and detachment.In one embodiment, if the codon optimized sequences are more effectivethan naturally-occurring sequences in humans, it is anticipated thatlower dosages stated above will be useful. Still other dosages in theseranges may be selected by the attending physician, taking into accountthe physical state of the subject, preferably human, being treated, theage of the subject, the particular ocular disorder (e.g., RDH12-mediateddisorder) and the degree to which the disorder, if progressive, hasdeveloped.

Yet another aspect described herein are various methods of treating anRDH12-mediated disorder or ocular disorder by administering the codonoptimized RDH12 DNA, vectors, viruses or other pharmaceuticalcompositions containing same. In another embodiment a method ofpreventing, treating, arresting progression of or ameliorating visionloss due to the above-described ocular diseases and retinal changesassociated therewith is provided. Still other methods are designed forrestoring partial or full vision or visual acuity to a subject with anocular disorder. Vision loss associated with LCA or RP refers to anydecrease in peripheral vision, central (reading) vision, night vision,day vision, loss of color perception, loss of contrast sensitivity, orreduction in visual acuity. Other vision problems that may be treatedusing the described methods include photophobia and nystagmus.

Generally, the methods include administering to a mammalian subject inneed thereof, an effective amount of a composition comprising arecombinant adeno-associated virus (AAV) carrying a nucleic acidsequence encoding a normal or functional RDH12 protein, or fragmentthereof, under the control of regulatory sequences which express theproduct of the gene in the subject's ocular cells, and apharmaceutically acceptable carrier.

In one embodiment, such a method is designed for treating, retarding orhalting progression of blindness in a mammalian subject having one ormore of the ocular diseases described above, such as LCA or RP. The rAAVcarrying the RDH12 optimized coding sequence or another functional RDH12sequence, preferably suspended in a physiologically compatible carrier,diluent, excipient and/or adjuvant, may be administered to a desiredsubject including without limitation, a cat, dog, or other non-humanmammalian subject. This method comprises administering to a subject inneed thereof any of the nucleic acid sequences, expression cassettes,rAAV genomes, plasmids, vectors or rAAV vectors or compositionscontaining them. In one embodiment, the composition is deliveredsubretinally. In another embodiment, the composition is deliveredintravitreally. In still another embodiment, the composition isdelivered using a combination of administrative routes suitable fortreatment of ocular diseases, and may also involve administration viathe palpebral vein or other intravenous or conventional administrationroutes.

For use in these methods, the volume and viral titer of each dosage isdetermined individually, as further described herein, and may be thesame or different from other treatments performed in the same, orcontralateral, eye. In another embodiment, a single, larger volumetreatment is made in order to treat the entire eye. The dosages,administrations and regimens may be determined by the attendingphysician given the teachings of this specification.

In one embodiment, the composition is administered in a single dosageselected from those above listed in a single affected eye. In anotherembodiment, the composition is administered as a single dosage selectedfrom those above listed in a both affected eyes, either simultaneouslyor sequentially. Sequential administration may imply a time gap ofadministration from one eye to another from intervals of minutes, hours,days, weeks or months. In another embodiment, the method involvesadministering the compositions to an eye two or more dosages (e.g.,split dosages).

In still other embodiments, the compositions described herein may bedelivered in a single composition or multiple compositions. Optionally,two or more different AAV may be delivered, or multiple viruses [see,e.g., WO 2011/126808 and WO 2013/049493]. In another embodiment,multiple viruses may contain different replication-defective viruses(e.g., AAV and adenovirus).

In certain embodiments of the invention it is desirable to performnon-invasive retinal imaging and functional studies to identify areas ofthe rod and cone photoreceptors to be targeted for therapy. In theseembodiments, clinical diagnostic tests are employed to determine theprecise location(s) for one or more subretinal injection(s). These testsmay include electroretinography (ERG), perimetry, topographical mappingof the layers of the retina and measurement of the thickness of itslayers by means of confocal scanning laser ophthalmoscopy (cSLO) andoptical coherence tomography (OCT), topographical mapping of conedensity via adaptive optics (AO), functional eye exam, etc, dependingupon the species of the subject being treated, their physical status andhealth and the dosage. In view of the imaging and functional studies, insome embodiments of the invention one or more injections are performedin the same eye in order to target different areas of the affected eye.The volume and viral titer of each injection is determined individually,as further described below, and may be the same or different from otherinjections performed in the same, or contralateral, eye. In anotherembodiment, a single, larger volume injection is made in order to treatthe entire eye. In one embodiment, the volume and concentration of therAAV composition is selected so that only the region of damaged rod andcone receptors is impacted. In another embodiment, the volume and/orconcentration of the rAAV composition is a greater amount, in orderreach larger portions of the eye, including non-damaged photoreceptors.

In one embodiment of the methods described herein, a one-timeintra-ocular delivery of a composition such as those described herein,e.g., an AAV delivery of an optimized RDH12 cassette, is useful inpreventing vision loss and blindness in millions of individuals affectedwith such ocular disorders or multi-systemic diseases without regard togenotype or environmental exposure.

Thus, in one embodiment, the composition is administered before diseaseonset. In another embodiment, the composition is administered prior tothe initiation of vision impairment or loss. In another embodiment, thecomposition is administered after initiation of vision impairment orloss. In yet another embodiment, the composition is administered whenless than 90% of the rod and/or cones or photoreceptors are functioningor remaining, as compared to a non-diseased eye.

In another embodiment, the method includes performing additionalstudies, e.g., functional and imaging studies to determine the efficacyof the treatment. For examination in animals, such tests include retinaland visual function assessment via electroretinograms (ERGs) looking atrod and cone photoreceptor function, optokinetic nystagmus,pupillometry, water maze testing, light-dark preference histology(retinal thickness, rows of nuclei in the outer nuclear layer,immunofluorescence to document transgene expression, cone photoreceptorcounting, staining of retinal sections with peanut agglutinin—whichidentifies cone photoreceptor sheaths). Other suitable tests of efficacyare sampling of anterior chamber fluid to document presence of the RDH12transgenic proteins.

Specifically for human subjects, following administration of a dosage ofa compositions described in this specification, the subject is testedfor efficacy of treatment using electroretinograms (ERGs) to examine rodand cone photoreceptor function, pupillometry visual acuity, contrastsensitivity color vision testing, visual field testing (Humphrey visualfields/Goldmann visual fields), perimetry mobility test (obstaclecourse), and reading speed test. Other useful post-treatment efficacytest to which the subject is exposed following treatment with apharmaceutical composition described herein are functional magneticresonance imaging (fMRI), full-field light sensitivity testing, retinalstructure studies including optical coherence tomography, fundusphotography, fundus autofluorescence, adaptive optics scanning, and/orlaser ophthalmoscopy. These and other efficacy tests are described inU.S. Pat. No. 8,147,82; in co-pending International patent applicationpublication WO 2014/011210 or WO 2014/124282, incorporated by reference.

In yet another embodiment, any of the above described methods isperformed in combination with another, or secondary, therapy. In stillother embodiments, the methods of treatment of these ocular diseasesinvolve treating the subject with the composition described in detailherein in combination with another therapy, such as antibiotictreatment, palliative treatment for pain, and the like. The additionaltherapy may be any now known, or as yet unknown, therapy which helpsprevent, arrest or ameliorate these mutations or defects or any of theeffects associated therewith. The secondary therapy can be administeredbefore, concurrent with, or after administration of the compositionsdescribed above. In one embodiment, a secondary therapy involvesnon-specific approaches for maintaining the health of the retinal cells,such as administration of neurotrophic factors, anti-oxidants,anti-apoptotic agents. The non-specific approaches are achieved throughinjection of proteins, recombinant DNA, recombinant viral vectors, stemcells, fetal tissue, or genetically modified cells. The latter couldinclude genetically modified cells that are encapsulated.

In another embodiment, the invention provides a method to prevent, orarrest photoreceptor function loss, or increase photoreceptor functionin the subject. Photoreceptor function may be assessed using thefunctional studies described above and in the examples below, e.g., ERGor perimetry, which are conventional in the art. As used herein“photoreceptor function loss” means a decrease in photoreceptor functionas compared to a normal, non-diseased eye or the same eye at an earliertime point. As used herein, “increase photoreceptor function” means toimprove the function of the photoreceptors or increase the number orpercentage of functional photoreceptors as compared to a diseased eye(having the same ocular disease), the same eye at an earlier time point,a non-treated portion of the same eye, or the contralateral eye of thesame patient.

In another aspect, the invention provides method of improvingphotoreceptor structure in the subject. As used herein “improvingphotoreceptor structure” refers (in the region of the retina that istreated) to one or more of an increase or decrease in outer nuclearlayer (ONL) thickness, or arresting progression of ONL thickening orthinning, across the entire retina, in the central retina, or theperiphery; increase or decrease in outer plexiform layer (OPL)thickness, or arresting progression of OPL thickening or thinning,across the entire retina, in the central retina, or the periphery;decrease in rod and cone inner segment (IS) shortening; decrease inshortening and loss of outer segments (OS); decrease in bipolar celldendrite retraction, or an increase in bipolar cell dendrite length oramount; and reversal of opsin mislocalization.

In another aspect, the invention provides a method of preventingRDH12-associated-LCA in a subject at risk of developing said disease.Subjects at risk of developing this ocular disorder include those with afamily history of LCA and those with one or more confirmed mutations inthe RDH12 gene.

For each of the described methods, the treatment may be used to preventthe occurrence of retinal damage or to rescue eyes having mild oradvanced disease. As used herein, the term “rescue” means to preventprogression of the disease to total blindness, prevent spread of damageto uninjured photoreceptor cells or to improve damage in injuredphotoreceptor cells. Thus, in one embodiment, the composition isadministered before disease onset. In another embodiment, thecomposition is administered after the initiation of opsinmislocalization. In another embodiment, the composition is administeredprior to the initiation of photoreceptor loss. In another embodiment,the composition is administered after initiation of photoreceptor loss.In yet another embodiment, the composition is administered when lessthan 90% of the photoreceptors are functioning or remaining, as comparedto a non-diseased eye. In another embodiment, the composition isadministered when less than 80% of the photoreceptors are functioning orremaining. In another embodiment, the composition is administered whenless than 70% of the photoreceptors are functioning or remaining. Inanother embodiment, the composition is administered when less than 60%of the photoreceptors are functioning or remaining. In anotherembodiment, the composition is administered when less than 50% of thephotoreceptors are functioning or remaining. In another embodiment, thecomposition is administered when less than 40% of the photoreceptors arefunctioning or remaining. In another embodiment, the composition isadministered when less than 30% of the photoreceptors are functioning orremaining. In another embodiment, the composition is administered whenless than 20% of the photoreceptors are functioning or remaining. Inanother embodiment, the composition is administered when less than 10%of the photoreceptors are functioning or remaining. In one embodiment,the composition is administered only to one or more regions of the eye,e.g., those which have retained photoreceptors. In another embodiment,the composition is administered to the entire eye.

In another embodiment, a method of treating or preventingRDH12-associated LCA or RP in a subject in need thereof is provided. Themethod includes identifying a subject having, or at risk of developing,RDH12-associated LCA or RP; performing genotypic analysis andidentifying at least one mutation in the RHD12 gene; performingnon-invasive retinal imaging and functional studies and identifyingareas of retained photoreceptors to be targeted for therapy; andadministering to the subject an effective concentration of acomposition, whereby RDH12-associated LCA or RP is prevented, arrestedor ameliorated. The composition includes a recombinant virus carrying anucleic acid sequence encoding a normal photoreceptor cell-specific geneunder the control of a promoter sequence which expresses the product ofthe gene in the photoreceptor cells, and a pharmaceutically acceptablecarrier. Genotypic analysis is routine in the art and may include theuse of PCR to identify one or more mutations in the nucleic acidsequence of the RDH12 gene. See, e.g., Meindl et al, Nat Gen, May 1996,13:35, Vervoort, R. et al, 2000. Nat Genet 25(4): 462-466 (cited above);and Vervoort, R. and Wright, A.F. 2002. Human Mutation 19: 486-500, eachof which is incorporated herein by reference.

The following examples disclose specific embodiments of the nucleic acidsequences, expression cassettes, rAAV genome and viral vectors for usein treating the ocular diseases specified herein. These specificembodiments illustrate various aspects of the invention. These examplesshould be construed to encompass any and all variations that becomeevident as a result of the teaching provided herein.

The examples below established proof-of concept of gene augmentationtherapy in RDH12 knock out (RDH12 −/−) mice and HEK293 cells. Expressionof codon optimized cDNA in HEK293 cells was shown to be 20% higher thanthe wildtype gene. Proviral plasmid backbones with modifications aredescribed, substantially as in WO2012/158,757, but with differentstuffer sequence (derived from phage lambda). The RDH12 coding sequenceis codon optimized, allowing for more efficient expression. In addition,we have included both native and codon optimized hRDH12 sequences in theplasmid design, and have shown efficacy results both in vitro and invivo.

The proviral plasmids generate proof-of-concept data, safety andpreclinical toxicity data and ultimately human clinical trials for RDH12patients. The invention described herein involves novel optimized cDNAsof Retinal Dehydrogenase 12 (RDH12) that encode a functional RDH12protein. In one embodiment, a codon optimized cDNA is designed fortreating LCA13. Transgene cassettes are optimized by genetic engineeringand because different sequences of nucleotides in a codon can encode thesame amino acid, one can alter the nucleotide sequence, but stillgenerate the same protein product. In other words, one can takeadvantage of multiple “synonymous” codons to generate the same proteinproduct.

Additional experiments are also being carried out in induced pluripotentstem cells (iPCs) derived from RDH12 patients. These data stronglysuggest clinical relevance and usefulness of the proviral plasmidspackaged in AAV vectors as a method to treat RDH12 induced ocularconditions. In addition, in vitro models such as HEK293 and iPS cellsare useful for testing the potency of the vectors.

EXAMPLE 1: CODON OPTIMIZED RDH12 SEQUENCE

The codon-optimized nucleic acid sequence encoding a functional RDH12SEQ ID NO: 3 was generated by modifying a nature human RDH12 sequence toadd a complete Kozak consensus at the 5′ end embedded in a NotI site andby adding WI and BamHI sites at the 3′ end (restriction sites forcloning). A TGA stop codon was embedded in the Bc1I site to facilitateoptimal epitope tagging. This embodiment also avoids the use of certainrestriction enzymes identified above.

The open reading frame (ORF) of codon optimized SEQ ID NO:3 differs fromthe native sequence by 22%, i.e., it shares only 78% identity withnative hRDH12, as shown in FIGS. 1A-1B.

EXAMPLE 2—CONSTRUCTION OF RDH12-EXPRESSING AAV

We generated adeno-associated virus proviral cis-plasmids containing thehuman native RDH12 cDNA with and without a myc tag based on the p618backbone (see U.S. Pat. No. 9,249,425, incorporated by reference hereinfor the sequences employed in p618). In these proviral plasmids, thehRDH12 cDNA is driven by the constitutive CMV.CβA promoter (CBAe), i.e.,pAAV.CMVe.native-hRDH12 and AAV.CBAe.h-native RDH12.myc.

As described in U.S. Pat. No. 9,249,425, the proviral plasmids alsocontain a 5′ AAV ITR sequence, the ITR flanked upstream by restrictionsite 1 and downstream by restriction site 2; (b) a promoter flankedupstream by restriction site 2 and downstream by restriction site 3; (c)a polylinker sequence comprising restriction site 3, restriction site 4and restriction site 5. In the embodiment described here, the transgenecomprising a codon optimized nucleic acid sequence that encodes RDH12 islocated between any two of the restriction sites 3, 4 and 5, withoutmodification thereof, wherein the transgene is operatively linked to,and under the regulatory control of, the promoter; (d) a polyadenylationsequence flanked upstream by restriction site 4 or 5 and downstream byrestriction site 6; and (e) a 3′ AAV ITR sequence flanked upstream byrestriction site 6 and downstream by restriction site 7. Eachrestriction site 1 through 7 occurs only once in the plasmid and iscleaved by a different enzyme that cannot cleave another restrictionsite in the plasmid. Restriction sites 1 through 7 are positioned topermit independent and repeated removal, replacement or substitution ofone or more of element (a), (b), (c), (d) and (e) or the entire AAVgenome (a) through (e) from the plasmid. Such a proviral plasmid furthercomprises a plasmid backbone comprising elements necessary forreplication in bacterial cells, and a resistance gene. In anotherembodiment, the plasmid backbone comprises one or more of (a) 5′ and 3′transcriptional terminator/insulator sequences that isolatetranscription in the backbone from transcription in the modularrecombinant AAV genome; or (b) a non-coding stuffer sequence thatincreases the backbone length and prevents reverse packaging ofnon-functional AAV genomes. These transgene cassettes are compatiblewith the cargo capacity of AAV vectors. The constructs were verified byrestriction mapping and DNA sequencing analysis.

See, e.g., the schematic drawings of AAV.CBAe.h-Native-RDH12,AAV.CBAe.h-Native RDH12-Myc and AAV.CBAe.h-codon opt- RDH12 of FIGS.10A-10C, respectively.

To confirm the expression of the wild type (native) human RDH12 proteinencoded in the proviral construct, COS-7 cells were transfected withpAAV-CMVe-native-hRDH12.myc. Transfected cells were subjected to immunefluorescence analysis and western blot analysis using antibodiesspecific to myc-tag.

The cells were then stained and examined electroscopically to showsuccessful transfection and the efficacy of gene transfer.Immunofluorescence analysis of cells transfected withpAAV.CMVe.native-hRDH12.myc demonstrated the expression of RDH12 proteinin transfected cells only. FIGS. 2A-2F shows 6 panels indicating theexpression of the RDH12 in the cells. FIGS. 2A-2B show untransfectedcontrol; FIGS. 2C-2D show RDH12Myc transfected cells; FIG. 2E showsRDH12Myc transfected cells; FIG. 2F is an enlargement of two cells fromFIG. 2D.

Western blot analysis further confirmed the expression of expected sizehuman RDH12 protein in transfected cells, with no band observed incontrol, untransfected cells. FIG. 2G is a gel showing RDH12Myctransfected COS-7 cells, control COS-7 cells, RDH12Myc transfected CHOcells and control CHO cells and two molecular weight markers. (FIG. 2G).

The pAAV.CMVe.native-hRDH12 and pAAV.CMVe.native-hRDH12.myc proviralplasmids were then used to produce recombinant AAV serotype vectors(AAV2, AAV8 and AAV7m8) using the triple transfection method involvingtransfection of subconfluent HEK293 cells by three plasmids: AAVcis-plasmid encoding the gene of interest, AAV trans-plasmid containingAAV rep and cap genes, and adenovirus helper plasmid, thereby generatingAAV8-RDH12, AAV2-RDH12 and AAV7m8-RDH12.

Briefly described, the AAV8-RDH12-myc or AAV7m8-RDH12-myc expressioncassettes are individually packaged in a selected AAV capsid byculturing a packaging cell carrying the plasmid in the presence ofsufficient viral sequences to permit packaging of the AAV genome into aninfectious AAV envelope or capsid. In one embodiment, a method forproducing the rAAV involves packaging in a stable rep and cap expressingmammalian host packaging cell line (such as B-50 as described inInternational Patent Application Publication No. WO 99/15685) with theadenovirus E1, E2a, and E4ORF6 DNA. Iodixanol gradient purification orgradient centrifugation is used to separate DNA containing viralparticles from the empty ones. This is followed by herparin-sepharoseagarose column chromatography. Vector titers are determined using aninfectious center assay. The vector genome is determined by silverstaining against an established reference lot. The purity of the vectorsis again examined by the clarity of a western gel. Virus preparationsare prepared in and combined to a desired total volume.

Still other methods of producing such rAAV particles involve use of aninsect cell packaging cell line, such as described in Smith et al, ref11, cited below.

The rAAV viral particles are suspended in a suitable excipient, such as180 mM NaCl, 10 mM NaPi, pH7.3, containing 0.0001% - 0.01% Pluronic F68(PF68). The composition of the saline component ranges from 160 mM to180 mM NaCl. Other buffers are useful in such compositions, includingHEPEs, sodium bicarbonate, TRIS, or 0.9% NaCl solution.

Several preparations of the rAAV are combined to a desired total volume.In one embodiment, a total volume is a dosage of 1×10¹¹ GC in a volumeof 300 microliters of buffer. In another embodiment, a total volume is adosage of 1×10⁸ GC in a volume of 300 microliters of buffer. In stillanother embodiment, a total volume is a dosage of 1×10⁶ to 1×10¹³ GC ina volume of 300 microliters of buffer. Contaminating helper adenovirusand native AAV, assayed by serial dilution cytopathic effect orinfectious center assay, respectively are anticipated to be less thanone or multiples orders of magnitude lower than vector AAV.

EXAMPLE 3—iPSCs AS A MODEL SYSTEM

Recent advances in patient-specific induced pluripotent stem cells(iPSCs) provide a suitable in vitro model system to study diseasepathogenesis. In order develop an in vitro model to study the functionof RDH12, iPS cells were generated from a human RDH12 patient andcharacterized. To confirm that infection with AAV2.CMV.CβA-native-hRDH12would result in the production of exogenous RDH12, we transducedaliquots of iPS cells with AAV2 CMV.CβA-native-hRDH12, with 1×10³,1×10⁴, 1×10⁵ or 2×10⁵, 3×10⁵ vector genomes (vg)/cell. Forty-eight hourslater, cell lysates were collected and analyzed via western blottingusing antibodies specific to myc tag. A clear dose-dependent productionof human RDH12 was observed in the transduced cell lysates. See theresults in FIG. 4.

In another in vitro experiment, recombinant virus AAV2-RDH12-Myc wasthen transfected into iPS cells at the indicated multiplicity oftransfection 1×10³ GC/cell, 1×10⁴ GC/cell, 1×10⁵ GC/cell, 2×10⁵ GC/cell,and 3×10⁵ GC/cell. A positive control is also shown. Expression isconfirmed as shown in FIG. 3.

EXAMPLE 4—LIGHT DAMAGE OF RDH12 KO ANIMALS

Rdh12^(−/−) mice with BALB/c background were obtained from Dr. AnneKasus-Jacobi, University of Oklahoma. These animals do not exhibit ahuman retinal degeneration phenotype when raised under normal cycliclight, but undergo degeneration after exposure to bright light.

To evaluate the efficacy of gene augmentation therapy in RDH12^(−/−)mice, the mice were pre-treated unilaterally with the experimental rAAVs(AAV8.CMV.CβA-native-hRDH12 or AAV7m8.CMV.CβA-native-hRDH12) anddetermined whether delivery of RDH12 protects these diseased retinasfrom light-induced degeneration. Animals at the age of 1-2 months wereinjected subretinally with AAV8.CMV.CβA-native-hRDH12 or intravitreallywith AAV7m8.CMV.CβA-native-hRDH12 unilaterally. Each rAAV was injectedin the right eye of a RDH12 KO mouse by injection of 10¹¹-10¹³ viralparticles or 10¹¹-10¹³ viral articles/ml buffer with the left eye leftuninfected. 3-4 weeks post injection, animals were exposed to a brightlight (10,000 Lux) for 4 h.

Effect of light in the retinas of these animals were evaluated byretinal function studies (electroretinograms, (ERGs)) before and 24 hafter light damage. Retina exposed to light should demonstrate evidenceof light damage. Animals at that point were subjected to 10 day recoveryperiod from light damage. We then measured the retinal function by ERGand the extent of photoreceptor cell death in retinal tissue sections.The mice were sacrificed and the eyes collected eyes for cryosectioning.Cryosectioned tissue is stained with Anti-myc antibody. Myc wasnon-specific. Nuclei were stained with DAPI.

FIGS. 5A and 5B show the a-wave amplitude differences in retinas ofRDH12^(−/−) mice injected either with AAV8 or AAV7m8.CMV.CβA-native-hRDH12 (AAV-RDH12) and untreated eyes.Electroretinography (ERG) revealed a partial functional rescue of thea-wave, which represents rod photoreceptor function, in AAV-RDH12treated retinas after light exposure. FIG. 5A shows a graph of an A-waveratio in RDH12.myc (AAV8-RDH12-Myc) injected vs. uninjected retina ofRDH12 KO mice (prebleach and post-bleach). FIG. 5B shows a graph of anA-wave prebleach and post-bleach ratio in RDH12.myc (AAV7m8-RDH12-Myc)injected vs. uninjected retina of RDH12 KO mice.

Vector-treated RDH12^(−/−) eyes were assessed for the degree ofhistological rescue that accompanied preservation of photoreceptorfunction after light damage (See FIGS. 6-9).

FIGS. 6A-6D shows experimental results for a single animal 136 in whichthe left eye (FIGS. 6A AND 6C) was uninjected. The right eye (FIGS. 6Band 6D) was injected with AAV7m8-RDH12-Myc. ERG baseline was performedfollowed by light damage, followed by a second ERG. The animals werehoused for 10 days and a third ERG was performed. Mice were sacrificedand eyes collected fixed and sectioned and stained with DAPI ((FIG. 6Aand 6B)) or with rhodopsin and DAPI ((FIG. 6C and 6D)).

FIG. 7A through 7C shows that the retinal architecture is preserved inan AAV7m8-RDH12-Myc injected retina compared to uninjected retina afterlight damage. FIG. 7A shows the left uninjected eye. FIG. 7B shows ahigher magnification image of the left eye, showing a thin ONL. FIG. 7Cis a right eye injected with AAV7m8-RDH12-Myc.

FIGS. 8A shows the retinal architecture of an animal with a left eyeuninjected showing a thin retina. FIG. 8B shows the animal's right eye,injected with AAV8-RDH12-Myc.

FIGS. 9A and 9B show the retinal architecture of a single animal 147with higher magnification images. FIG. 9A shows uninjected left eye.FIG. 9B shows right eye injected with AAV8-RDH12-Myc.

At low magnification, it is apparent that most treated RDH12^(−/−)retinas maintained a relatively normal ONL, whereas in the untreated eyeof the same mouse, the ONL contained few photoreceptor cell bodies inthe central retinal region. Higher magnification images showed that atypical treated retina retained a substantial Outer nuclear thicknessand outer segments. In contrast, in untreated eye from the same mouse,only one to three rows of ONL nuclei remained, with residual outersegments in the central retina.

The same tests are performed to confirm that the optimization of codonusage within the RDH12 gene results in increased levels of transgeneexpression and a better rescue of retinal degeneration. Codonoptimization is anticipated to reduce the viral dose needed foreffective reconstitution of RDH12.

EXAMPLE 5—EFFICACY IN HUMAN SUBJECTS

The rAAV particles are also employed to transduce cells of humansubject's retina after administration by subretinal injection of10¹⁰-10¹² GC or viral particles in a suspension in a suitable bufferedcarrier. Expression of codon optimized hRDH12 in transduced cells orretinas is assessed by retinal and visual function.

These functions are examined in humans using one or more of thetechniques: electroretinograms (ERGs) looking at rod and conephotoreceptor function pupillometry visual acuity contrast sensitivitycolor vision testing visual field testing (Humphrey visualfields/Goldmann visual fields) perimetry mobility test (obstacle course)reading speed test. Other useful tests include functional magneticresonance imaging (fMRI) full-field light sensitivity testing, retinalstructure studies including optical coherence tomography, fundusphotography, fundus autofluorescence, adaptive optics and scanning laserophthalmoscopy.

TABLE 1 (Sequence Listing Free Text) The following information isprovided for sequences containing free text under numeric identifier<223>. SEQ ID NO: (containing free text) Free Text Under <223> 3 Codonoptimized RDH12 DNA sequence 4 Translated functional RDH12 proteinsequence from codon optimized nucleic acid sequence

Each and every patent, patent application, and publication citedthroughout the specification is incorporated herein by reference. U.S.Provisional Patent Application No. 62/359,777, filed Jul. 8, 2016, andInternational Patent Application No. PCT/US2017/041122, filed Jul. 7,2017, are incorporated herein by reference. Similarly, the SEQ ID NOswhich are referenced herein and which appear in the appended SequenceListing are incorporated by reference. While the invention has beendescribed with reference to particular embodiments, it will beappreciated that modifications can be made without departing from thespirit of the invention. Such modifications are intended to fall withinthe scope of the appended claims.

1. A method of treating Leber Congenital Amaurosis (LCA) due to one ormore loss-of function mutations in the gene encoding the RetinolDehydrogenase 12 (RDH12) protein in a human subject, the methodcomprising administering into the subretinal space of at least one eyeof the subject an adeno-associated viral (AAV) vector comprising anucleic acid, wherein the nucleic acid comprises human RDH12 DNA andwherein the human RDH12 DNA encodes a protein comprising SEQ ID NO: 2,and wherein the AAV vector is AAV2/5.
 2. The method of claim 1, whereinthe RDH12 DNA is under the expression control of a human rhodopsinkinase 1 (hGRK1) promoter.
 3. The method of claim 1, wherein the humanRDH12 DNA is at least 70% identical to the full length of SEQ ID NO: 1.4. The method of claim 1, wherein the human RDH12 DNA is at least 90%identical to the full length of SEQ ID NO:
 1. 5. The method of claim 1,wherein the human RDH12 DNA is at least 95% identical to the full lengthof SEQ ID NO:
 1. 6. The method of claim 1, wherein the human RDH12 DNAis codon optimized for expression in a human cell.
 7. The method ofclaim 1, wherein a micro injection device is inserted into thesubretinal space.
 8. The method of claim 1, comprising administering thenucleic acid in an amount of about 10¹¹-10¹³ viral particles/mL.